Selective antisense compounds and uses thereof

ABSTRACT

Disclosed are oligomeric compounds which are useful for hybridizing to a complementary nucleic acid, including but not limited, to nucleic acids in a cell. The hybridization results in modulation of the amount activity or expression of the target nucleic acid in a cell.

FIELD OF THE INVENTION

The present invention pertains generally to chemically-modifiedoligonucleotides for use in research, diagnostics, and/or therapeutics.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0099WOSEQ.txt, created Aug. 1, 2012 which is 304 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Antisense compounds have been used to modulate target nucleic acids.Antisense compounds comprising a variety of chemical modifications andmotifs have been reported. In certain instances, such compounds areuseful as research tools, diagnostic reagents, and as therapeuticagents. In certain instances antisense compounds have been shown tomodulate protein expression by binding to a target messenger RNA (mRNA)encoding the protein. In certain instances, such binding of an antisensecompound to its target mRNA results in cleavage of the mRNA. Antisensecompounds that modulate processing of a pre-mRNA have also beenreported. Such antisense compounds alter splicing, interfere withpolyadenlyation or prevent formation of the 5′-cap of a pre-mRNA.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides oligomericcompounds comprising oligonucleotides. In certain embodiments, sucholigonucleotides comprise a region having a gapmer motif. In certainembodiments, such oligonucleotides consist of a region having a gapmermotif.

The present disclosure provides the following non-limiting numberedembodiments:

Embodiment 1

-   -   A oligomeric compound comprising a modified oligonucleotide        consisting of 10 to 30 linked nucleosides, wherein the modified        oligonucleotide has a modification motif comprising: a 5′-region        consisting of 2-8 linked 5′-region nucleosides, each        independently selected from among a modified nucleoside and an        unmodified deoxynucleoside, provided that at least one 5′-region        nucleoside is a modified nucleoside and wherein the 3′-most        5′-region nucleoside is a modified nucleoside;    -   a 3′-region consisting of 2-8 linked 3′-region nucleosides, each        independently selected from among a modified nucleoside and an        unmodified deoxynucleoside, provided that at least one 3′-region        nucleoside is a modified nucleoside and wherein the 5′-most        3′-region nucleoside is a modified nucleoside; and    -   a central region between the 5′-region and the 3′-region        consisting of 6-12 linked central region nucleosides, each        independently selected from among: a modified nucleoside and an        unmodified deoxynucleoside, wherein the 5′-most central region        nucleoside is an unmodified deoxynucleoside and the 3′-most        central region nucleoside is an unmodified deoxynucleoside;    -   wherein the modified oligonucleotide has a nucleobase sequence        complementary to the nucleobase sequence of a target region of a        target nucleic acid.

Embodiment 2

-   -   The oligomeric compound of embodiment 1, wherein the nucleobase        sequence of the target region of the target nucleic acid differs        from the nucleobase sequence of at least one non-target nucleic        acid by 1-3 differentiating nucleobases.

Embodiment 3

-   -   The oligomeric compound of embodiment 1, the nucleobase sequence        of the target region of the target nucleic acid differs from the        nucleobase sequence of at least one non-target nucleic acid by a        single differentiating nucleobase.

Embodiment 4

-   -   The oligomeric compound of embodiment 2 or 3, wherein the target        nucleic acid and the non-target nucleic acid are alleles of the        same gene.

Embodiment 5

-   -   The oligomeric compound of embodiment 4, wherein the single        differentiating nucleobase is a single-nucleotide polymorphism.

Embodiment 6

-   -   The oligomeric compound of embodiment 5, wherein the        single-nucleotide polymorphism is associated with a disease.

Embodiment 7

-   -   The oligomeric compound of embodiment 6, wherein the disease is        selected from among: Alzheimer's disease, Creutzfeldt-Jakob        disease, fatal familial insomnia, Alexander disease, Parkinson's        disease, amyotrophic lateral sclerosis, dentato-rubral and        pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion        dystonia, cardiomyopathy, chronic obstructive pulmonary disease        (COPD), liver disease, hepatocellular carcinoma, systemic lupus        erythematosus, hypercholesterolemia, breast cancer, asthma, Type        1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal        and bulbar muscular atrophy, Kennedy's disease, progressive        childhood posterior subcapsular cataracts, cholesterol gallstone        disease, arthrosclerosis, cardiovascular disease, primary        hypercalciuria, alpha-thallasemia, obsessive compulsive        disorder, Anxiety, comorbid depression, congenital visual        defects, hypertension, metabolic syndrome, prostate cancer,        congential myasthenic syndrome, peripheral arterial disease,        atrial fibrillation, sporadic pheochromocytoma, congenital        malformations, Machado-Joseph disease, Huntington's disease, and        Autosomal Dominant Retinitis Pigmentosa disease.

Embodiment 8

-   -   The oligomeric compound of embodiment 6, wherein the        single-nucleotide polymorphism is selected from among:        rs6446723, rs3856973, rs2285086, rs363092, rs916171, rs6844859,        rs7691627, rs4690073, rs2024115, rs11731237, rs362296,        rs10015979, rs7659144, rs363096, rs362273, rs16843804, rs362271,        rs362275, rs3121419, rs362272, rs3775061, rs34315806, rs363099,        rs2298967, rs363088, rs363064, rs363102, rs2798235, rs363080,        rs363072, rs363125, rs362303, rs362310, rs10488840, rs362325,        rs35892913, rs363102, rs363096, rs11731237, rs10015979,        rs363080, rs2798235, rs1936032, rs2276881, rs363070, rs35892913,        rs12502045, rs6446723, rs7685686, rs3733217, rs6844859, and        rs362331.

Embodiment 9

-   -   The oligomeric compound of embodiment 8, wherein the        single-nucleotide polymorphism is selected from among:        rs7685686, rs362303 rs4690072 and rs363088

Embodiment 10

-   -   The oligomeric compound of embodiment 2 or 3, wherein the target        nucleic acid and the non-target nucleic acid are transcripts        from different genes.

Embodiment 11

-   -   The oligomeric compound of any of embodiments 1-10, wherein the        3′-most 5′-region nucleoside comprises a bicyclic sugar moiety.

Embodiment 12

-   -   The oligomeric compound of embodiment 11, wherein the 3′-most        5′-region nucleoside comprises a cEt sugar moiety.

Embodiment 13

-   -   The oligomeric compound of embodiment 11, wherein the 3′-most        5′-region nucleoside comprises an LNA sugar moiety.

Embodiment 14

-   -   The oligomeric compound of any of embodiments 1-13, wherein the        central region consists of 6-10 linked nucleosides.

Embodiment 15

-   -   The oligomeric compound of any of embodiments 1-14, wherein the        central region consists of 6-9 linked nucleosides.

Embodiment 16

-   -   The oligomeric compound of embodiment 15, wherein the central        region consists of 6 linked nucleosides.

Embodiment 17

-   -   The oligomeric compound of embodiment 15, wherein the central        region consists of 7 linked nucleosides.

Embodiment 18

-   -   The oligomeric compound of embodiment 15, wherein the central        region consists of 8 linked nucleosides.

Embodiment 19

-   -   The oligomeric compound of embodiment 15, wherein the central        region consists of 9 linked nucleosides.

Embodiment 20

-   -   The oligomeric compound of any of embodiments 1-19, wherein each        central region nucleoside is an unmodified deoxynucleoside.

Embodiment 21

-   -   The oligomeric compound of any of embodiments 1-19, wherein at        least one central region nucleoside is a modified nucleoside.

Embodiment 22

-   -   The oligomeric compound of embodiment 21, wherein one central        region nucleoside is a modified nucleoside and each of the other        central region nucleosides is an unmodified deoxynucleoside.

Embodiment 23

-   -   The oligomeric compound of embodiment 21, wherein two central        region nucleosides are modified nucleosides and each of the        other central region nucleosides is an unmodified        deoxynucleoside.

Embodiment 24

-   -   The oligomeric compound of any of embodiments 21-23 wherein at        least one modified central region nucleoside is an RNA-like        nucleoside.

Embodiment 25

-   -   The oligomeric compound of any of embodiments 21-23 comprising        at least one modified central region nucleoside comprising a        modified sugar moiety.

Embodiment 26

-   -   The oligomeric compound of any of embodiments 21-25 comprising        at least one modified central region nucleoside comprising a        5′-methyl-2′-deoxy sugar moiety.

Embodiment 27

-   -   The oligomeric compound of any of embodiments 21-26 comprising        at least one modified central region nucleoside comprising a        bicyclic sugar moiety.

Embodiment 28

-   -   The oligomeric compound of any of embodiments 21-27 comprising        at least one modified central region nucleoside comprising a cEt        sugar moiety.

Embodiment 29

-   -   The oligomeric compound of any of embodiments 21-28 comprising        at least one modified central region nucleoside comprising an        LNA sugar moiety.

Embodiment 30

-   -   The oligomeric compound of any of embodiments 21-29 comprising        at least one modified central region nucleoside comprising an        α-LNA sugar moiety.

Embodiment 31

-   -   The oligomeric compound of any of embodiments 21-29 comprising        at least one modified central region nucleoside comprising a        2′-substituted sugar moiety.

Embodiment 32

-   -   The oligomeric compound of embodiment 31 wherein at least one        modified central region nucleoside comprises a 2′-substituted        sugar moiety comprising a 2′ substituent selected from among:        halogen, optionally substituted allyl, optionally substituted        amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O,        S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or        N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3,        O—(CH2)2-O—N(Rm)(Rn) or O—CH2-C(═O)—N(Rm)(Rn), where each Rm and        Rn is, independently, H, an amino protecting group or        substituted or unsubstituted C₁-C₁₀ alkyl;    -   wherein each optionally substituted group is optionally        substituted with a substituent group independently selected from        among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro        (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,        alkenyl and alkynyl.

Embodiment 33

-   -   The oligomeric compound of embodiment 32 wherein at least one        modified central region nucleoside comprises a 2′-substituted        sugar moiety comprising a 2′ substituent selected from among: a        halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂,        OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃,        O(CH₂)₃—N(R₁)(R₂), O(CH₂)₂—ON(R₁)(R₂),        O(CH₂)₂—O(CH₂)₂—N(R₁)(R₂), OCH₂C(═O)—N(R₁)(R₂),        OCH₂C(═O)—N(R₃)—(CH₂)₂—N(R₁)(R₂), and        O(CH₂)₂—N(R₃)—C(═NR₄)[N(R₁)(R₂)]; wherein R₁, R₂, R₃ and R₄ are        each, independently, H or C₁-C₆ alkyl.

Embodiment 34

-   -   The oligomeric compound of embodiment 33 wherein the 2′        substituent is selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃ (MOE),        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 35

-   -   The oligomeric compound of any of embodiments 21-34 comprising        at least one modified central region nucleoside comprising a        2′-MOE sugar moiety.

Embodiment 36

-   -   The oligomeric compound of any of embodiments 21-35 comprising        at least one modified central region nucleoside comprising a        2′-OMe sugar moiety.

Embodiment 37

-   -   The oligomeric compound of any of embodiments 21-36 comprising        at least one modified central region nucleoside comprising a        2′-F sugar moiety.

Embodiment 38

-   -   The oligomeric compound of any of embodiments 21-37 comprising        at least one modified central region nucleoside comprising a        2′-(ara)-F sugar moiety.

Embodiment 39

-   -   The oligomeric compound of any of embodiments 21-38 comprising        at least one modified central region nucleoside comprising a        sugar surrogate.

Embodiment 40

-   -   The oligomeric compound of embodiment 39 comprising at least one        modified central region nucleoside comprising an F-HNA sugar        moiety.

Embodiment 41

-   -   The oligomeric compound of embodiment 39 or 40 comprising at        least one modified central region nucleoside comprising an HNA        sugar moiety.

Embodiment 42

-   -   The oligomeric compound of any of embodiments 21-41 comprising        at least one modified central region nucleoside comprising a        modified nucleobase.

Embodiment 43

-   -   The oligomeric compound of embodiment 42 comprising at least one        modified central region nucleoside comprising a modified        nucleobase selected from a 2-thio pyrimidine and a 5-propyne        pyrimidine.

Embodiment 44

-   -   The oligomeric compound of any of embodiments 21-43, wherein the        2^(nd) nucleoside from the 5′-end of the central region is a        modified nucleoside.

Embodiment 45

-   -   The oligomeric compound of any of embodiments 21-44, wherein the        3^(rd) nucleoside from the 5′-end of the central region is a        modified nucleoside.

Embodiment 46

-   -   The oligomeric compound of any of embodiments 21-45, wherein the        4^(th) nucleoside from the 5′-end of the central region is a        modified nucleoside.

Embodiment 47

-   -   The oligomeric compound of any of embodiments 21-46, wherein the        5^(th) nucleoside from the 5′-end of the central region is a        modified nucleoside.

Embodiment 48

-   -   The oligomeric compound of any of embodiments 21-47, wherein the        6^(th) nucleoside from the 5′-end of the central region is a        modified nucleoside.

Embodiment 49

-   -   The oligomeric compound of any of embodiments 21-48, wherein the        8^(th) nucleoside from the 3′-end of the central region is a        modified nucleoside.

Embodiment 50

-   -   The oligomeric compound of any of embodiments 21-49, wherein the        7^(th) nucleoside from the 3′-end of the central region is a        modified nucleoside.

Embodiment 51

-   -   The oligomeric compound of any of embodiments 21-50, wherein the        6^(th) nucleoside from the 3′-end of the central region is a        modified nucleoside.

Embodiment 52

-   -   The oligomeric compound of any of embodiments 21-51, wherein the        5^(th) nucleoside from the 3′-end of the central region is a        modified nucleoside.

Embodiment 53

-   -   The oligomeric compound of any of embodiments 21-52, wherein the        4^(th) nucleoside from the 3′-end of the central region is a        modified nucleoside.

Embodiment 54

-   -   The oligomeric compound of any of embodiments 21-53, wherein the        3^(rd) nucleoside from the 3′-end of the central region is a        modified nucleoside.

Embodiment 55

-   -   The oligomeric compound of any of embodiments 21-54, wherein the        2^(nd) nucleoside from the 3′-end of the central region is a        modified nucleoside.

Embodiment 56

-   -   The oligomeric compound of any of embodiments 21-55, wherein the        modified nucleoside is a 5′-methyl-2′-deoxy sugar moiety.

Embodiment 57

-   -   The oligomeric compound of any of embodiments 21-55, wherein the        modified nucleoside is a 2-thio pyrimidine.

Embodiment 58

-   -   The oligomeric compound of any of embodiments 21-55, wherein the        central region comprises no region having more than 4 contiguous        unmodified deoxynucleosides.

Embodiment 59

-   -   The oligomeric compound of any of embodiments 21-55, wherein the        central region comprises no region having more than 5 contiguous        unmodified deoxynucleosides.

Embodiment 60

-   -   The oligomeric compound of any of embodiments 21-55, wherein the        central region comprises no region having more than 6 contiguous        unmodified deoxynucleosides.

Embodiment 61

-   -   The oligomeric compound of any of embodiments 21-55, wherein the        central region comprises no region having more than 7 contiguous        unmodified deoxynucleosides.

Embodiment 62

-   -   The oligomeric compound of any of embodiments 1-14 or 21-59,        wherein the central region has a nucleoside motif selected from        among: DDDDDDDDDD, DDDDXDDDDD; DDDDDXDDDDD; DDDXDDDDD;        DDDDXDDDDDD; DDDDXDDDD; DDXDDDDDD; DDDXDDDDDD; DXDDDDDD;        DDXDDDDDDD; DDXDDDDD; DDXDDDXDDD; DDDXDDDXDDD; DXDDDXDDD;        DDXDDDXDD; DDXDDDDXDDD; DDXDDDDXDD; DXDDDDXDDD; DDDDXDDD;        DDDXDDD; DXDDDDDDD; DDDDXXDDD; and DXXDXXDXX; wherein    -   each D is an unmodified deoxynucleoside; and each X is a        modified nucleoside.

Embodiment 63

-   -   The oligomeric compound of any of embodiments 1-14 or 21-59,        wherein the central region has a nucleoside motif selected from        among: DDDDDDDDD; DXDDDDDDD; DDXDDDDDD; DDDXDDDDD; DDDDXDDDD;        DDDDDXDDD; DDDDDDXDD; DDDDDDDXD; DXXDDDDDD; DDDDDDXXD;        DDXXDDDDD; DDDXXDDDD; DDDDXXDDD; DDDDDXXDD; DXDDDDDXD;        DXDDDDXDD; DXDDDXDDD; DXDDXDDDD; DXDXDDDDD; DDXDDDDXD;        DDXDDDXDD; DDXDDXDDD; DDXDXDDDD; DDDXDDDXD; DDDXDDXDD;        DDDXDXDDD; DDDDXDDXD; DDDDXDXDD; and DDDDDXDXD wherein each D is        an unmodified deoxynucleoside; and each X is a modified        nucleoside.

Embodiment 64

-   -   The oligomeric compound of any of embodiments 1-14 or 21-59,        wherein the central region has a nucleoside motif selected from        among: DDDDXDDDD, DXDDDDDDD, DXXDDDDDD, DDXDDDDDD, DDDXDDDDD,        DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, and DDDDDDDXD.

Embodiment 65

-   -   The oligomeric compound of any of embodiments 1-14 or 21-59,        wherein the central region has a nucleoside motif selected from        among: DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD,        DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD,        DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD,        DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD.

Embodiment 66

-   -   The oligomeric compound of any of embodiments 1-14 or 21-59,        wherein the central region has a nucleoside motif selected from        among: DDDDDDD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD,        DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD,        DDDXXDD, DDDXDXD, and DDDDXXD.

Embodiment 67

-   -   The oligomeric compound of any of embodiments 1-14 or 21-59,        wherein the central region has a nucleoside motif selected from        among: DDDDDD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD,        DXDDXD, DDXXDD, DDXDXD, and DDDXXD.

Embodiment 68

-   -   The oligomeric compound of any of embodiments 1-14 or 21-59,        wherein the central region has a nucleoside motif selected from        among: DDDDDD, DDDDDDD, DDDDDDDD, DDDDDDDDD, DXDDDD, DDXDDD,        DDDXDD, DDDDXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD,        DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD,        DXDDDDDDD; DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD,        DDDDDDXDD, DDDDDDDXD, DXDDDDDDDD, DDXDDDDDDD, DDDXDDDDDD,        DDDDXDDDDD, DDDDDXDDDD, DDDDDDXDDD, DDDDDDDXDD, and DDDDDDDDXD.

Embodiment 69

-   -   The oligomeric compound of embodiments 62-68, wherein each X        comprises a modified nucleobase.

Embodiment 70

-   -   The oligomeric compound of embodiments 62-68, wherein each X        comprises a modified sugar moiety.

Embodiment 71

-   -   The oligomeric compound of embodiments 62-68, wherein each X        comprises 2-thio-thymidine.

Embodiment 72

-   -   The oligomeric compound of embodiments 62-68, wherein each X        nucleoside comprises an F-HNA sugar moiety.

Embodiment 73

-   -   The oligomeric compound of embodiments 62-68, wherein the        nucleobase sequence of the target region of the target nucleic        acid differs from the nucleobase sequence of at least one        non-target nucleic acid by a single differentiating nucleobase,        and wherein the location of the single differentiating        nucleobase is represented by X.

Embodiment 74

-   -   The oligomeric compound of embodiment 73, wherein the target        nucleic acid and the non-target nucleic acid are alleles of the        same gene.

Embodiment 75

-   -   The oligomeric compound of embodiment 73, wherein the single        differentiating nucleobase is a single-nucleotide polymorphism.

Embodiment 76

-   -   The oligomeric compound of any of embodiments 1-75, wherein the        5′ region consists of 2 linked 5′-region nucleosides.

Embodiment 77

-   -   The oligomeric compound of any of embodiments 1-75, wherein the        5′ region consists of 3 linked 5′-region nucleosides.

Embodiment 78

-   -   The oligomeric compound of any of embodiments 1-75, wherein the        5′ region consists of 4 linked 5′-region nucleosides.

Embodiment 79

-   -   The oligomeric compound of any of embodiments 1-75, wherein the        5′ region consists of 5 linked 5′-region nucleosides.

Embodiment 80

-   -   The oligomeric compound of any of embodiments 1-75, wherein the        5′ region consists of 6 linked 5′-region nucleosides.

Embodiment 81

-   -   The oligomeric compound of any of embodiments 1-80, wherein at        least one 5′-region nucleoside is an unmodified deoxynucleoside.

Embodiment 82

-   -   The oligomeric compound of any of embodiments 1-80, wherein each        5′-region nucleoside is a modified nucleoside.

Embodiment 83

-   -   The oligomeric compound of any of embodiments 1-80 wherein at        least one 5′-region nucleoside is an RNA-like nucleoside.

Embodiment 84

-   -   The oligomeric compound of any of embodiments 1-80 wherein each        5′-region nucleoside is an RNA-like nucleoside.

Embodiment 85

-   -   The oligomeric compound of any of embodiments 1-80 comprising at        least one modified 5′-region nucleoside comprising a modified        sugar.

Embodiment 86

-   -   The oligomeric compound of embodiment 80 comprising at least one        modified 5′-region nucleoside comprising a bicyclic sugar        moiety.

Embodiment 87

-   -   The oligomeric compound of embodiment 86 comprising at least one        modified 5′-region nucleoside comprising a cEt sugar moiety.

Embodiment 88

-   -   The oligomeric compound of embodiment 85 or 86 comprising at        least one modified 5′-region nucleoside comprising an LNA sugar        moiety.

Embodiment 89

-   -   The oligomeric compound of any of embodiments 76-80 comprising        of at least one modified 5′-region nucleoside comprising a        2′-substituted sugar moiety.

Embodiment 90

-   -   The oligomeric compound of embodiment 89 wherein at least one        modified central region nucleoside comprises a 2′-substituted        sugar moiety comprising a 2′ substituent selected from among:        halogen, optionally substituted allyl, optionally substituted        amino, azido, optionally substituted SH, CN, OCN, CF₃, OCF₃, O,        S, or N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or        N(R_(m))-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH₂)₂SCH₃,        O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where        each R_(m) and R_(n) is, independently, H, an amino protecting        group or substituted or unsubstituted C₁-C₁₀ alkyl;    -   wherein each optionally substituted group is optionally        substituted with a substituent group independently selected from        among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro        (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,        alkenyl and alkynyl.

Embodiment 91

-   -   The oligomeric compound of embodiment 90 wherein at least one        modified 5′-region nucleoside comprises a 2′-substituted sugar        moiety comprising a 2′-substituent selected from among: a        halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂,        OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃ (MOE), O(CH₂)₂—SCH₃,        O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁)(R₂), O(CH₂)₂—ON(R₁)(R₂),        O(CH₂)₂—O(CH₂)₂—N(R₁)(R₂), OCH₂C(═O)—N(R₁)(R₂),        OCH₂C(═O)—N(R₃)—(CH₂)₂—N(R₁)(R₂), and        O(CH₂)₂—N(R₃)—C(═NR₄)[N(R₁)(R₂)]; wherein R₁, R₂, R₃ and R₄ are        each, independently, H or C₁-C₆ alkyl.

Embodiment 92

-   -   The oligomeric compound of embodiment 91, wherein the        2′-substituent is selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 93

-   -   The oligomeric compound of any of embodiments 89-92 comprising        at least one modified 5′-region nucleoside comprising a 2′-MOE        sugar moiety.

Embodiment 94

-   -   The oligomeric compound of any of embodiments 89-92 comprising        at least one modified 5′-region nucleoside comprising a 2′-OMe        sugar moiety.

Embodiment 95

-   -   The oligomeric compound of any of embodiments 89-92 comprising        at least one modified 5′-region nucleoside comprising a 2′-F        sugar moiety.

Embodiment 96

-   -   The oligomeric compound of any of embodiments 89-92 comprising        at least one modified 5′-region nucleoside comprising a        2′-(ara)-F sugar moiety.

Embodiment 97

-   -   The oligomeric compound of any of embodiments 82-96 comprising        of at least one modified 5′-region nucleoside comprising a sugar        surrogate.

Embodiment 98

-   -   The oligomeric compound of embodiment 97 comprising at least one        modified 5′-region nucleoside comprising an F-HNA sugar moiety.

Embodiment 99

-   -   The oligomeric compound of embodiment 97 or 98 comprising at        least one modified 5′-region nucleoside comprising an HNA sugar        moiety.

Embodiment 100

-   -   The oligomeric compound of any of embodiments 1-99 comprising at        least one modified 5′-region nucleoside comprising a modified        nucleobase.

Embodiment 101

-   -   The oligomeric compound of embodiment 100, wherein the modified        nucleoside comprises 2-thio-thymidine.

Embodiment 102

-   -   The oligomeric compound of any of embodiments 1-101, wherein the        5′-region has a motif selected from among:    -   ADDA; ABDAA; ABBA; ABB; ABAA; AABAA; AAABAA; AAAABAA; AAAAABAA;        AAABAA; AABAA; ABAB; ABADB; ABADDB; AAABB; AAAAA; ABBDC; ABDDC;        ABBDCC; ABBDDC; ABBDCC; ABBC; AA; AAA; AAAA; AAAAB; AAAAAAA;        AAAAAAAA; ABBB; AB; ABAB; AAAAB; AABBB; AAAAB; and AABBB,        wherein each A is a modified nucleoside of a first type, each B        is a modified nucleoside of a second type, each C is a modified        nucleoside of a third type, and each D is an unmodified        deoxynucleoside.

Embodiment 103

-   -   The oligomeric compound of any of embodiments 1-101, wherein the        5′-region has a motif selected from among:    -   AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW,        ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB,        AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, BBBBAA, and        AAABW wherein each A is a modified nucleoside of a first type,        each B is a modified nucleoside of a second type, and each W is        a modified nucleoside of a third type.

Embodiment 104

-   -   The oligomeric compound of any of embodiments 1-101, wherein the        5′-region has a motif selected from among: ABB; ABAA; AABAA;        AAABAA; ABAB; ABADB; AAABB; AAAAA; AA; AAA; AAAA; AAAAB; ABBB;        AB; and ABAB, wherein each A is a modified nucleoside of a first        type, each B is a modified nucleoside of a second type, and each        W is a modified nucleoside of a third type.

Embodiment 105

-   -   The oligomeric compound of embodiments 102-104, wherein each A        nucleoside comprises a 2′-substituted sugar moiety.

Embodiment 106

-   -   The oligomeric compound of embodiment 105 wherein at least one        central region nucleoside comprises a 2′-substituted sugar        moiety comprising a 2′ substituent selected from among: halogen,        optionally substituted allyl, optionally substituted amino,        azido, optionally substituted SH, CN, OCN, CF₃, OCF₃, O, S, or        N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or        N(R_(m))-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH₂)₂SCH₃,        O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where        each R_(m) and R_(n) is, independently, H, an amino protecting        group or substituted or unsubstituted C₁-C₁₀ alkyl; wherein each        optionally substituted group is optionally substituted with a        substituent group independently selected from among: hydroxyl,        amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol,        thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 107

-   -   The oligomeric compound of embodiment 102-106, wherein each A        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 108

-   -   The oligomeric compound of embodiment 107, wherein each A        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: F, OCH₃, O(CH₂)₂—OCH₃.

Embodiment 109

-   -   The oligomeric compound of embodiments 102-106, wherein each A        nucleoside comprises a bicyclic sugar moiety.

Embodiment 110

-   -   The oligomeric compound of embodiment 109, wherein each A        nucleoside comprises a bicyclic sugar moiety selected from        among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.

Embodiment 111

-   -   The oligomeric compound of any of embodiments 102-110, wherein        each A comprises a modified nucleobase.

Embodiment 112

-   -   The oligomeric compound of embodiment 111, wherein each A        comprises a modified nucleobase selected from among a 2-thio        pyrimidine and a 5-propyne pyrimidine.

Embodiment 113

-   -   The oligomeric compound of embodiment 112, wherein each A        comprises 2-thio-thymidine.

Embodiment 114

-   -   The oligomeric compound of embodiment 102-106, wherein each A        nucleoside comprises an unmodified 2′-deoxyfuranose sugar        moiety.

Embodiment 115

-   -   The oligomeric compound of embodiment 102-106, wherein each A        nucleoside comprises an F-HNA sugar moiety.

Embodiment 116

-   -   The oligomeric compound of any of embodiments 102-115, wherein        each B nucleoside comprises a 2′-substituted sugar moiety.

Embodiment 117

-   -   The oligomeric compound of embodiment 116, wherein at least one        central region nucleoside comprises a 2′-substituted sugar        moiety comprising a 2′ substituent selected from among: halogen,        optionally substituted allyl, optionally substituted amino,        azido, optionally substituted SH, CN, OCN, CF₃, OCF₃, O, S, or        N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or        N(R_(m))-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH₂)₂SCH₃,        O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where        each R_(m) and R_(n) is, independently, H, an amino protecting        group or substituted or unsubstituted C₁-C₁₀ alkyl; wherein each        optionally substituted group is optionally substituted with a        substituent group independently selected from among: hydroxyl,        amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol,        thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 118

-   -   The oligomeric compound of embodiment 117, wherein each B        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 119

-   -   The oligomeric compound of embodiment 118, wherein each B        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: F, OCH₃, O(CH₂)₂—OCH₃.

Embodiment 120

-   -   The oligomeric compound of any of embodiments 102-115, wherein        each B nucleoside comprises a bicyclic sugar moiety.

Embodiment 121

-   -   The oligomeric compound of embodiment 120, wherein each B        nucleoside comprises a bicyclic sugar moiety selected from        among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.

Embodiment 122

-   -   The oligomeric compound of any of embodiments 102-115, wherein        each B comprises a modified nucleobase.

Embodiment 123

-   -   The oligomeric compound of embodiment 122, wherein each B        comprises a modified nucleobase selected from among a 2-thio        pyrimidine and a 5-propyne pyrimidine.

Embodiment 124

-   -   The oligomeric compound of embodiment 123, wherein each B        comprises 2-thio-thymidine.

Embodiment 125

-   -   The oligomeric compound of embodiment 102-106, wherein each B        nucleoside comprises an unmodified 2′-deoxyfuranose sugar        moiety.

Embodiment 126

-   -   The oligomeric compound of embodiment 102-115, wherein each B        nucleoside comprises an F-HNA sugar moiety.

Embodiment 127

-   -   The oligomeric compound of any of embodiments 102-126, wherein        each C nucleoside comprises a 2′-substituted sugar moiety.

Embodiment 128

-   -   The oligomeric compound of embodiment 127, wherein at least one        central region nucleoside comprises a 2′-substituted sugar        moiety comprising a 2′ substituent selected from among: halogen,        optionally substituted allyl, optionally substituted amino,        azido, optionally substituted SH, CN, OCN, CF₃, OCF₃, O, S, or        N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or        N(R_(m))-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH₂)₂SCH₃,        O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where        each R_(m) and R_(n) is, independently, H, an amino protecting        group or substituted or unsubstituted C₁-C₁₀ alkyl; wherein each        optionally substituted group is optionally substituted with a        substituent group independently selected from among: hydroxyl,        amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol,        thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 129

-   -   The oligomeric compound of embodiment 128, wherein each C        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 130

-   -   The oligomeric compound of embodiment 129, wherein each C        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: F, OCH₃, O(CH₂)₂—OCH₃.

Embodiment 131

-   -   The oligomeric compound of any of embodiments 102-126, wherein        each C nucleoside comprises a bicyclic sugar moiety.

Embodiment 132

-   -   The oligomeric compound of embodiment 131, wherein each C        nucleoside comprises a bicyclic sugar moiety selected from        among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.

Embodiment 133

-   -   The oligomeric compound of any of embodiments 102-126, wherein        each C comprises a modified nucleobase.

Embodiment 134

-   -   The oligomeric compound of embodiment 133, wherein each C        comprises a modified nucleobase selected from among a 2-thio        pyrimidine and a 5-propyne pyrimidine.

Embodiment 135

-   -   The oligomeric compound of embodiment 134, wherein each C        comprises 2-thio-thymidine.

Embodiment 136

-   -   The oligomeric compound of embodiment 102-126, wherein each C        comprises an F—HNA sugar moiety.

Embodiment 137

-   -   The oligomeric compound of embodiment 102-126, wherein each C        nucleoside comprises an unmodified 2′-deoxyfuranose sugar        moiety.

Embodiment 138

-   -   The oligomeric compound of any of embodiments 102-138, wherein        each W nucleoside comprises a 2′-substituted sugar moiety.

Embodiment 139

-   -   The oligomeric compound of embodiment 138, wherein at least one        central region nucleoside comprises a 2′-substituted sugar        moiety comprising a 2′ substituent selected from among: halogen,        optionally substituted allyl, optionally substituted amino,        azido, optionally substituted SH, CN, OCN, CF₃, OCF₃, O, S, or        N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or        N(R_(m))-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH₂)₂SCH₃,        O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where        each R_(m) and R_(n) is, independently, H, an amino protecting        group or substituted or unsubstituted C₁-C₁₀ alkyl; wherein each        optionally substituted group is optionally substituted with a        substituent group independently selected from among: hydroxyl,        amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol,        thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 140

-   -   The oligomeric compound of embodiment 139, wherein each W        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 141

-   -   The oligomeric compound of embodiment 139, wherein each W        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: F, OCH₃, O(CH₂)₂—OCH₃.

Embodiment 142

-   -   The oligomeric compound of any of embodiments 102-137, wherein        each W nucleoside comprises a bicyclic sugar moiety.

Embodiment 143

-   -   The oligomeric compound of embodiment 142, wherein each W        nucleoside comprises a bicyclic sugar moiety selected from        among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.

Embodiment 144

-   -   The oligomeric compound of any of embodiments 102-137, wherein        each W comprises a modified nucleobase.

Embodiment 145

-   -   The oligomeric compound of embodiment 144, wherein each W        comprises a modified nucleobase selected from among a 2-thio        pyrimidine and a 5-propyne pyrimidine.

Embodiment 146

-   -   The oligomeric compound of embodiment 145, wherein each W        comprises 2-thio-thymidine.

Embodiment 147

-   -   The oligomeric compound of embodiment 102-137, wherein each W        comprises an F—HNA sugar moiety.

Embodiment 148

-   -   The oligomeric compound of embodiment 102-137, wherein each W        nucleoside comprises an unmodified 2′-deoxyfuranose sugar        moiety.

Embodiment 149

-   -   The oligomeric compound of any of embodiments 1-148, wherein the        3′ region consists of 2 linked 3′-region nucleosides.

Embodiment 150

-   -   The oligomeric compound of any of embodiments 1-148, wherein the        3′ region consists of 3 linked 3′-region nucleosides.

Embodiment 151

-   -   The oligomeric compound of any of embodiments 1-148, wherein the        3′ region consists of 4 linked 3′-region nucleosides.

Embodiment 152

-   -   The oligomeric compound of any of embodiments 1-148, wherein the        3′ region consists of 5 linked 3′-region nucleosides.

Embodiment 153

-   -   The oligomeric compound of any of embodiments 1-148, wherein the        3′ region consists of 6 linked 3′-region nucleosides.

Embodiment 154

-   -   The oligomeric compound of any of embodiments 1-153, wherein at        least one 3′-region nucleoside is an unmodified deoxynucleoside.

Embodiment 155

-   -   The oligomeric compound of any of embodiments 1-154, wherein        each 3′-region nucleoside is a modified nucleoside.

Embodiment 156

-   -   The oligomeric compound of any of embodiments 1-153, wherein at        least one 3′-region nucleoside is an RNA-like nucleoside.

Embodiment 157

-   -   The oligomeric compound of any of embodiments 1-154, wherein        each 3′-region nucleoside is an RNA-like nucleoside.

Embodiment 158

-   -   The oligomeric compound of any of embodiments 1-153, comprising        at least one modified 3′-region nucleoside comprising a modified        sugar.

Embodiment 159

-   -   The oligomeric compound of embodiment 158, comprising at least        one modified 3′-region nucleoside comprising a bicyclic sugar        moiety.

Embodiment 160

-   -   The oligomeric compound of embodiment 159, comprising at least        one modified 3′-region nucleoside comprising a cEt sugar moiety.

Embodiment 161

-   -   The oligomeric compound of embodiment 159, comprising at least        one modified 3′-region nucleoside comprising an LNA sugar        moiety.

Embodiment 162

-   -   The oligomeric compound of any of embodiments 1-162 comprising        of at least one modified 3′-region nucleoside comprising a        2′-substituted sugar moiety.

Embodiment 163

-   -   The oligomeric compound of embodiment 162, wherein at least one        central region nucleoside comprises a 2′-substituted sugar        moiety comprising a 2′ substituent selected from among: halogen,        optionally substituted allyl, optionally substituted amino,        azido, optionally substituted SH, CN, OCN, CF₃, OCF₃, O, S, or        N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or        N(R_(m))-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH₂)₂SCH₃,        O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where        each R_(m) and R_(n) is, independently, H, an amino protecting        group or substituted or unsubstituted C₁-C₁₀ alkyl; wherein each        optionally substituted group is optionally substituted with a        substituent group independently selected from among: hydroxyl,        amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol,        thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 164

-   -   The oligomeric compound of embodiment 163 wherein at least one        modified 3′-region nucleoside comprises a 2′-substituted sugar        moiety comprising a 2′-substituent selected from among: a        halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂,        OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃ (MOE), O(CH₂)₂—SCH₃,        O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁)(R₂), O(CH₂)₂—ON(R₁)(R₂),        O(CH₂)₂—O(CH₂)₂—N(R₁)(R₂), OCH₂C(═O)—N(R₁)(R₂),        OCH₂C(═O)—N(R₃)—(CH₂)₂—N(R₁)(R₂), and        O(CH₂)₂—N(R₃)—C(═NR₄)[N(R₁)(R₂)]; wherein R₁, R₂, R₃ and R₄ are        each, independently, H or C₁-C₆ alkyl.

Embodiment 165

-   -   The oligomeric compound of embodiment 164, wherein the        2′-substituent is selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 166

-   -   The oligomeric compound of any of embodiments 162-165 comprising        at least one modified 3′-region nucleoside comprising a 2′-MOE        sugar moiety.

Embodiment 167

-   -   The oligomeric compound of any of embodiments 162-166 comprising        at least one modified 3′-region nucleoside comprising a 2′-OMe        sugar moiety.

Embodiment 168

-   -   The oligomeric compound of any of embodiments 162-167 comprising        at least one modified 3′-region nucleoside comprising a 2′-F        sugar moiety.

Embodiment 169

-   -   The oligomeric compound of any of embodiments 162-168 comprising        at least one modified 3′-region nucleoside comprising a        2′-(ara)-F sugar moiety.

Embodiment 170

-   -   The oligomeric compound of any of embodiments 162-169 comprising        of at least one modified 3′-region nucleoside comprising a sugar        surrogate.

Embodiment 171

-   -   The oligomeric compound of embodiment 170 comprising at least        one modified 3′-region nucleoside comprising an F-HNA sugar        moiety.

Embodiment 172

-   -   The oligomeric compound of embodiment 170 comprising at least        one modified 3′-region nucleoside comprising an HNA sugar        moiety.

Embodiment 173

-   -   The oligomeric compound of any of embodiments 1-172 comprising        at least one modified 3′-region nucleoside comprising a modified        nucleobase.

Embodiment 174

-   -   The oligomeric compound of any of embodiments 1-173, wherein        each A comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: F, OCH₃, O(CH₂)₂—OCH₃, and        each B comprises a bicyclic sugar moiety selected from among:        LNA and cEt.

Embodiment 175

-   -   The oligomeric compound of embodiment 174, wherein each A        comprises O(CH₂)₂—OCH₃ and each B comprises cEt.

Embodiment 176

-   -   The oligomeric compound of any of embodiments 1-175, wherein the        3′-region has a motif selected from among: ABB, ABAA, AAABAA,        AAAAABAA, AABAA, AAAABAA, AAABAA, ABAB, AAAAA, AAABB, AAAAAAAA,        AAAAAAA, AAAAAA, AAAAB, AAAA, AAA, AA, AB, ABBB, ABAB, AABBB,        wherein each A is a modified nucleoside of a first type, each B        is a modified nucleoside of a second type.

Embodiment 177

-   -   The oligomeric compound of embodiments 1-175, wherein the        3′-region has a motif selected from among: ABB; AAABAA; AABAA;        AAAABAA; AAAAA; AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AB;        ABBB; and ABAB, wherein each A is a modified nucleoside of a        first type, each B is a modified nucleoside of a second type.

Embodiment 178

-   -   The oligomeric compound of embodiments 1-175, wherein the        3′-region has a motif selected from among: BBA, AAB, AAA, BBB,        BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB,        BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB,        BAAAA, and ABBBB, wherein each A is a modified nucleoside of a        first type, each B is a modified nucleoside of a second type,        and each W is a modified nucleoside of a first type, a second        type, or a third type.

Embodiment 179

-   -   The oligomeric compound of embodiments 176-178, wherein each A        nucleoside comprises a 2′-substituted sugar moiety.

Embodiment 180

-   -   The oligomeric compound of embodiments 176-178, wherein at least        one central region nucleoside comprises a 2′-substituted sugar        moiety comprising a 2′ substituent selected from among: halogen,        optionally substituted allyl, optionally substituted amino,        azido, optionally substituted SH, CN, OCN, CF₃, OCF₃, O, S, or        N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or        N(R_(m))-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH₂)₂SCH₃,        O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where        each R_(m) and R_(n) is, independently, H, an amino protecting        group or substituted or unsubstituted C₁-C₁₀ alkyl;    -   wherein each optionally substituted group is optionally        substituted with a substituent group independently selected from        among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro        (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,        alkenyl and alkynyl.

Embodiment 181

-   -   The oligomeric compound of embodiment 180, wherein each A        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 182

-   -   The oligomeric compound of embodiment 181, wherein each A        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: F, OCH₃, O(CH₂)₂—OCH₃.

Embodiment 183

-   -   The oligomeric compound of embodiments 176-178, wherein each A        nucleoside comprises a bicyclic sugar moiety.

Embodiment 184

-   -   The oligomeric compound of embodiment 183, wherein each A        nucleoside comprises a bicyclic sugar moiety selected from        among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.

Embodiment 185

-   -   The oligomeric compound of any of embodiments 176-178, wherein        each B nucleoside comprises a 2′-substituted sugar moiety.

Embodiment 186

-   -   The oligomeric compound of embodiment 185, wherein at least one        modified central region nucleoside comprises a 2′-substituted        sugar moiety comprising a 2′ substituent selected from among:        halogen, optionally substituted allyl, optionally substituted        amino, azido, optionally substituted SH, CN, OCN, CF₃, OCF₃, O,        S, or N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or        N(R_(m))-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH₂)₂SCH₃,        O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where        each R_(m) and R_(n) is, independently, H, an amino protecting        group or substituted or unsubstituted C₁-C₁₀ alkyl;    -   wherein each optionally substituted group is optionally        substituted with a substituent group independently selected from        among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro        (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,        alkenyl and alkynyl.

Embodiment 187

-   -   The oligomeric compound of embodiment 185, wherein each B        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 188

-   -   The oligomeric compound of embodiment 187, wherein each B        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: F, OCH₃, O(CH₂)₂—OCH₃.

Embodiment 189

-   -   The oligomeric compound of any of embodiments 176-178, wherein        each B nucleoside comprises a bicyclic sugar moiety.

Embodiment 190

-   -   The oligomeric compound of embodiment 189, wherein each B        nucleoside comprises a bicyclic sugar moiety selected from        among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.

Embodiment 191

-   -   The oligomeric compound of any of embodiments 176-190, wherein        each A comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: F, OCH₃, O(CH₂)₂—OCH₃, and        each B comprises a bicyclic sugar moiety selected from among:        LNA and cEt.

Embodiment 192

-   -   The oligomeric compound of embodiment 191, wherein each A        comprises O(CH₂)₂—OCH₃ and each B comprises cEt.

Embodiment 193

-   -   The oligomeric compound of any of embodiments 176-192, wherein        each W nucleoside comprises a 2′-substituted sugar moiety.

Embodiment 194

-   -   The oligomeric compound of embodiment 193, wherein at least one        central region nucleoside comprises a 2′-substituted sugar        moiety comprising a 2′ substituent selected from among: halogen,        optionally substituted allyl, optionally substituted amino,        azido, optionally substituted SH, CN, OCN, CF₃, OCF₃, O, S, or        N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or        N(R_(m))-alkynyl; optionally substituted O-alkylenyl-O-alkyl,        optionally substituted alkynyl, optionally substituted alkaryl,        optionally substituted aralkyl, optionally substituted        O-alkaryl, optionally substituted O-aralkyl, O(CH₂)₂SCH₃,        O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where        each R_(m) and R_(n) is, independently, H, an amino protecting        group or substituted or unsubstituted C₁-C₁₀ alkyl; wherein each        optionally substituted group is optionally substituted with a        substituent group independently selected from among: hydroxyl,        amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol,        thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 195

-   -   The oligomeric compound of embodiment 193, wherein each W        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: a halogen, OCH₃, OCF₃,        OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.

Embodiment 196

-   -   The oligomeric compound of embodiment 195, wherein each W        nucleoside comprises a 2′-substituted sugar moiety comprising a        2′-substituent selected from among: F, OCH₃, O(CH₂)₂—OCH₃.

Embodiment 197

-   -   The oligomeric compound of any of embodiments 176-192, wherein        each W nucleoside comprises a bicyclic sugar moiety.

Embodiment 198

-   -   The oligomeric compound of embodiment 197, wherein each W        nucleoside comprises a bicyclic sugar moiety selected from        among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.

Embodiment 199

-   -   The oligomeric compound of any of embodiments 176-192, wherein        each W comprises a modified nucleobase.

Embodiment 200

-   -   The oligomeric compound of embodiment 199, wherein each W        comprises a modified nucleobase selected from among a 2-thio        pyrimidine and a 5-propyne pyrimidine.

Embodiment 201

-   -   The oligomeric compound of embodiment 200, wherein each W        comprises 2-thio-thymidine.

Embodiment 202

-   -   The oligomeric compound of embodiment 176-192, wherein each W        comprises an F-HNA sugar moiety.

Embodiment 203

-   -   The oligomeric compound of embodiment 202, wherein each W        nucleoside comprises an unmodified 2′-deoxyfuranose sugar        moiety.

Embodiment 204

-   -   The oligomeric compound of embodiments 1-203, wherein the        5′-region has a motif selected from among: AB, ABB, AAA, BBB,        BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB,        ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB,        ABBBB, BBBBB, AAABW, AAAAA, and BBBBAA;        -   wherein the 3′-region has a motif selected from among: BBA,            AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB,            BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA,            AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB;        -   wherein the central region has a nucleoside motif selected            from among: DDDDDD, DDDDDDD, DDDDDDDD, DDDDDDDDD,            DDDDDDDDDD, DXDDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD,            DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXXDDDDDD, DDDDDDXXD,            DDXXDDDDD, DDDXXDDDD, DDDDXXDDD, DDDDDXXDD, DXDDDDDXD,            DXDDDDXDD, DXDDDXDDD, DXDDXDDDD, DXDXDDDDD, DDXDDDDXD,            DDXDDDXDD, DDXDDXDDD, DDXDXDDDD, DDDXDDDXD, DDDXDDXDD,            DDDXDXDDD, DDDDXDDXD, DDDDXDXDD, and DDDDDXDXD, DDDDDDDD,            DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD,            DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD,            DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD,            DDDDXXDD, DDDDXDXD, and DDDDDXXD, DXDDDDD, DDXDDDD, DDDXDDD,            DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD,            DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD,            DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD,            DDXXDD, DDXDXD, and DDDXXD; and        -   wherein each A is a modified nucleoside of a first type,            each B is a modified nucleoside of a second type, each W is            a modified nucleoside of a first type, a second type, or a            third type, each D is an unmodified deoxynucleoside, and            each X is a modified nucleoside or a modified nucleobase.

Embodiment 205

-   -   The oligomeric compound of embodiment 204, wherein the 5′-region        has a motif selected from among:    -   AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, ABBW, ABBWW,        ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB,        AAAAB, AABBB, ABBBB, BBBBB, AAABW, and BBBBAA; and wherein the        3′-region has a BBA motif.

Embodiment 206

-   -   The oligomeric compound of embodiment 204 or 205, wherein one of        A or B comprises a bicyclic sugar moiety, another of A or B        comprises a 2′-MOE sugar moiety, and W comprises a        2-thio-thymidine nucleobase.

Embodiment 207

-   -   The oligomeric compound of embodiment 204 or 205, wherein one of        A or B comprises a bicyclic sugar moiety, another of A or B        comprises a 2′-MOE sugar moiety, and W comprises FHNA.

Embodiment 208

-   -   The oligomeric compound of embodiment 204 or 205, wherein one of        A or B comprises cEt, another of A or B comprises a 2′-modified        sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

Embodiment 209

-   -   The oligomeric compound of embodiment 204 or 205, wherein one of        A or B comprises cEt, another of A or B comprises a 2′-modified        sugar moiety, and W comprises FHNA.

Embodiment 210

-   -   The oligomeric compound of embodiment 204 or 205, wherein each A        comprises MOE, each B comprises cEt, and each W is selected from        among cEt or FHNA.

Embodiment 211

-   -   The oligomeric compound of embodiment 204 or 205, wherein each W        comprises cEt.

Embodiment 212

-   -   The oligomeric compound of embodiment 204 or 205, wherein each W        comprises 2-thio-thymidine.

Embodiment 213

-   -   The oligomeric compound of embodiment 204 or 205, wherein each W        comprises FHNA.

Embodiment 214

-   -   The oligomeric compound of any of embodiments 1-213 comprising        at least one modified internucleoside linkage.

Embodiment 215

-   -   The oligomeric compound of embodiment 214, wherein each        internucleoside linkage is a modified internucleoside linkage.

Embodiment 216

-   -   The oligomeric compound of embodiment 214 or 215 comprising at        least one phosphorothioate internucleoside linkage.

Embodiment 217

-   -   The oligomeric compound of any of embodiments 214 or 215        comprising at least one methylphosphonate internucleoside        linkage.

Embodiment 218

-   -   The oligomeric compound of any of embodiments 214 or 215        comprising one methylphosphonate internucleoside linkage.

Embodiment 219

-   -   The oligomeric compound of any of embodiments 214 or 215        comprising two methylphosphonate internucleoside linkages.

Embodiment 220

-   -   The oligomeric compound of embodiment 217, wherein at least one        of the 3^(rd), 4^(th), 5^(th), 6^(th) and/or, 7^(th)        internucleoside from the 5′-end is a methylphosphonate        internucleoside linkage.

Embodiment 221

-   -   The oligomeric compound of embodiment 217, wherein at least one        of the 3^(rd), 4^(th), 5^(th), 6^(th) and/or 7^(th)        internucleoside from the 3′-end is a methylphosphonate        internucleoside linkage.

Embodiment 222

-   -   The oligomeric compound of embodiment 217, wherein at least one        of the 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th), 8^(th), 9^(th),        10^(th), 11^(th), and/or 12^(th) internucleoside from the 5′-end        is a methylphosphonate internucleoside linkage, and wherein at        least one of the 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th), 8^(th),        9^(th), 10^(th), 11^(th), and/or 12^(th) internucleoside from        the 5′-end is a modified nucleoside.

Embodiment 223

-   -   The oligomeric compound of embodiment 217, wherein at least one        of the 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th), 8^(th), 9^(th),        10^(th), 11^(th), and/or 12^(th) internucleoside from the 3′-end        is a methylphosphonate internucleoside linkage, and wherein at        least one of the 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th), 8^(th),        9^(th), 10^(th), 11^(th), and/or 12^(th) internucleoside from        the 3′-end is a modified nucleoside.

Embodiment 224

-   -   The oligomeric compound of any of embodiments 1-223 comprising        at least one conjugate group.

Embodiment 225

-   -   The oligomeric compound of embodiment 1-223, wherein the        conjugate group consists of a conjugate.

Embodiment 226

-   -   The oligomeric compound of embodiment 225, wherein the conjugate        group consists of a conjugate and a conjugate linker.

Embodiment 227

-   -   The oligomeric compound of any of embodiments 1-226, wherein the        nucleobase sequence of the modified oligonucleotide is 100%        complementary to the nucleobase sequence of the target region of        the target nucleic acid.

Embodiment 228

-   -   The oligomeric compound of any of embodiments 1-226, wherein the        nucleobase sequence of the modified oligonucleotide contains one        mismatch relative to the nucleobase sequence of the target        region of the target nucleic acid.

Embodiment 229

-   -   The oligomeric compound of any of embodiments 1-226, wherein the        nucleobase sequence of the modified oligonucleotide contains two        mismatches relative to the nucleobase sequence of the target        region of the target nucleic acid.

Embodiment 230

-   -   The oligomeric compound of any of embodiments 1-226, wherein the        nucleobase sequence of the modified oligonucleotide comprises a        hybridizing region and at least one non-targeting region,        wherein the nucleobase sequence of the hybridizing region is        complementary to the nucleobase sequence of the target region of        the target nucleic acid.

Embodiment 231

-   -   The oligomeric compound of embodiment 230, wherein the        nucleobase sequence of the hybridizing region is 100%        complementary to the nucleobase sequence of the target region of        the target nucleic acid.

Embodiment 232

-   -   The oligomeric compound of embodiment 230, wherein the        nucleobase sequence of the hybridizing region contains one        mismatched relative to the nucleobase sequence of the target        region of the target nucleic acid.

Embodiment 233

-   -   The oligomeric compound of any of embodiments 230-232, wherein        the nucleobase sequence of at least one non-targeting region is        complementary to a portion of the hybridizing region of the        modified oligonucleotide.

Embodiment 234

-   -   The oligomeric compound of embodiment 233, wherein the        nucleobase sequence of at least one non-targeting region is 100%        complementary to a portion of the hybridizing region of the        modified oligonucleotide.

Embodiment 235

-   -   The oligomeric compound of embodiment 1-234 wherein the        nucleobase sequence of the modified oligonucleotide comprises        two non-targeting regions flanking a central hybridizing region.

Embodiment 236

-   -   The oligomeric compound of embodiment 235, wherein the two        non-targeting regions are complementary to one another.

Embodiment 237

-   -   The oligomeric compound of embodiment 236, wherein the two        non-targeting regions are 100% complementary to one another.

Embodiment 238

-   -   The oligomeric compound of any of embodiments 2-237, wherein the        nucleobase sequence of the modified oligonucleotide aligns with        the nucleobase of the target region of the target nucleic acid        such that a distinguishing nucleobase of the target region of        the target nucleic acid aligns with a target-selective        nucleoside within the central region of the modified        oligonucleotide.

Embodiment 239

-   -   The oligomeric compound of any of embodiments 3-237, wherein the        nucleobase sequence of the modified oligonucleotide aligns with        the nucleobase of the target region of the target nucleic acid        such that the single distinguishing nucleobase of the target        region of the target nucleic acid aligns with a target-selective        nucleoside within the central region of the modified        oligonucleotide.

Embodiment 240

-   -   The oligomeric compound of embodiment 238 or 239, wherein the        target-selective nucleoside is the 5′-most nucleoside of the        central region.

Embodiment 241

-   -   The oligomeric compound of embodiment 238 or 239, wherein the        target-selective nucleoside is the 2^(nd) nucleoside from the        5′-end of the central region.

Embodiment 242

-   -   The oligomeric compound of embodiment 238 or 239, wherein the        target-selective nucleoside is at the 3^(rd) nucleoside from the        5′-end of the central region.

Embodiment 243

-   -   The oligomeric compound of embodiment 238 or 239, wherein the        target-selective nucleoside is at the 5^(th) nucleoside from the        5′-end of the central region.

Embodiment 244

-   -   The oligomeric compound of embodiment 238 or 239, wherein the        target-selective nucleoside is at the 7^(th) nucleoside from the        5′-end of the central region.

Embodiment 245

-   -   The oligomeric compound of embodiment 238 or 239, wherein the        target-selective nucleoside is at the 9^(th) nucleoside from the        5′-end of the central region.

Embodiment 246

-   -   The oligomeric compound of any of embodiments 238 or 239, or        241-245, wherein the target-selective nucleoside is at the        2^(nd) nucleoside from the 3′-end of the central region.

Embodiment 247

-   -   The oligomeric compound of any of embodiments 238 or 239, or        241-245, wherein the target-selective nucleoside is at the        5^(th) nucleoside from the 3′-end of the central region.

Embodiment 248

-   -   The oligomeric compound of any of embodiments 1-247, wherein        target-selective nucleoside is an unmodified deoxynucleoside.

Embodiment 249

-   -   The oligomeric compound of any of embodiments 1-247, wherein        target-selective nucleoside is a modified nucleoside.

Embodiment 250

-   -   The oligomeric compound of embodiment 249, wherein the        target-selective nucleoside is a sugar modified nucleoside.

Embodiment 251

-   -   The oligomeric compound of embodiment 250, wherein the        target-selective nucleoside comprises a sugar modification        selected from among: 2′-MOE, 2′-F, 2′-(ara)-F, HNA, FHNA, cEt,        and α-L-LNA.

Embodiment 252

-   -   The oligomeric compound of any of embodiments 1-251, wherein the        target-selective nucleoside comprises a nucleobase modification.

Embodiment 253

-   -   The oligomeric compound of embodiment 252, wherein the modified        nucleobase is selected from among: a 2-thio pyrimidine and a        5-propyne pyrimidine.

Embodiment 254

-   -   The oligomeric compound of any of embodiments 1-253, wherein the        oligomeric compound is an antisense compound.

Embodiment 255

-   -   The oligomeric compound of embodiment 254, wherein the        oligomeric compound selectively reduces expression of the target        relative to the non-target.

Embodiment 256

-   -   The oligomeric compound of embodiment 255, wherein the        oligomeric compound reduces expression of target at least        two-fold more than it reduces expression of the non-target.

Embodiment 257

-   -   The oligomeric compound of embodiment 256, having an EC₅₀ for        reduction of expression of target that is at least least        two-fold lower than its EC₅₀ for reduction of expression of the        non-target, when measured in cells.

Embodiment 258

-   -   The oligomeric compound of embodiment 256, having an ED₅₀ for        reduction of expression of target that is at least least        two-fold lower than its ED₅₀ for reduction of expression of the        non-target, when measured in an animal.

Embodiment 259

-   -   The oligomeric compound of embodiments 1-10, having an        E-E-E-K-K-(D)₇-E-E-K motif, wherein each E is a 2′-MOE        nucleoside and each K is a cEt nucleoside.

Embodiment 260

-   -   A method comprising contacting a cell with an oligomeric        compound of any of embodiments 1-259.

Embodiment 261

-   -   The method of embodiment 260, wherein the cell is in vitro.

Embodiment 262

-   -   The method of embodiment 260, wherein the cell is in an animal.

Embodiment 263

-   -   The method of embodiment 262, wherein the animal is a human.

Embodiment 264

-   -   The method of embodiment 263, wherein the animal is a mouse.

Embodiment 265

-   -   A pharmaceutical composition comprising an oligomeric compound        of any of embodiments 1-259 and a pharmaceutically acceptable        carrier or diluent.

Embodiment 266

-   -   A method of administering a pharmaceutical composition of        embodiment 265 to an animal.

Embodiment 267

-   -   The method of embodiment 266, wherein the animal is a human.

Embodiment 268

-   -   The method of embodiment 266, wherein the animal is a mouse.

Embodiment 269

-   -   Use of an oligomeric compound of any of embodiments 1-259 for        the preparation of a medicament for the treatment or        amelioration of Alzheimer's disease, Creutzfeldt-Jakob disease,        fatal familial insomnia, Alexander disease, Parkinson's disease,        amyotrophic lateral sclerosis, dentato-rubral and        pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion        dystonia, cardiomyopathy, chronic obstructive pulmonary disease        (COPD), liver disease, hepatocellular carcinoma, systemic lupus        erythematosus, hypercholesterolemia, breast cancer, asthma, Type        1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal        and bulbar muscular atrophy, Kennedy's disease, progressive        childhood posterior subcapsular cataracts, cholesterol gallstone        disease, arthrosclerosis, cardiovascular disease, primary        hypercalciuria, alpha-thallasemia, obsessive compulsive        disorder, Anxiety, comorbid depression, congenital visual        defects, hypertension, metabolic syndrome, prostate cancer,        congential myasthenic syndrome, peripheral arterial disease,        atrial fibrillation, sporadic pheochromocytoma, congenital        malformations, Machado-Joseph disease, Huntington's disease, and        Autosomal Dominant Retinitis Pigmentosa disease.

Embodiment 270

-   -   A method of ameliorating a symptom of Alzheimer's disease,        Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander        disease, Parkinson's disease, amyotrophic lateral sclerosis,        dentato-rubral and pallido-luysian atrophy DRPA,        spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy,        chronic obstructive pulmonary disease (COPD), liver disease,        hepatocellular carcinoma, systemic lupus erythematosus,        hypercholesterolemia, breast cancer, asthma, Type 1 diabetes,        Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar        muscular atrophy, Kennedy's disease, progressive childhood        posterior subcapsular cataracts, cholesterol gallstone disease,        arthrosclerosis, cardiovascular disease, primary hypercalciuria,        alpha-thallasemia, obsessive compulsive disorder, Anxiety,        comorbid depression, congenital visual defects, hypertension,        metabolic syndrome, prostate cancer, congential myasthenic        syndrome, peripheral arterial disease, atrial fibrillation,        sporadic pheochromocytoma, congenital malformations,        Machado-Joseph disease, Huntington's disease, and Autosomal        Dominant Retinitis Pigmentosa disease, comprising administering        an oligomeric compound of any of embodiments 1-259 to an animal        in need thereof.

Embodiment 271

-   -   The method of embodiment 270, wherein the animal is a human.

Embodiment 272

-   -   The method of embodiment 270, wherein the animal is a mouse.

In certain embodiments, including but not limited to any of the abovenumbered embodiments, oligomeric compounds including oligonucleotidesdescribed herein are capable of modulating expression of a target RNA.In certain embodiments, the target RNA is associated with a disease ordisorder, or encodes a protein that is associated with a disease ordisorder. In certain embodiments, the oligomeric compounds oroligonucleotides provided herein modulate the expression of function ofsuch RNA to alleviate one or more symptom of the disease or disorder.

In certain embodiments, oligomeric compounds including oligonucleotidesdescribe herein are useful in vitro. In certain embodiments sucholigomeric compounds are used in diagnostics and/or for targetvalidation experiments.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

A. DEFINITIONS

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21^(st) edition, 2005; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosure areincorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Chemicalmodifications of oligonucleotides include nucleoside modifications(including sugar moiety modifications and nucleobase modifications) andinternucleoside linkage modifications. In reference to anoligonucleotide, chemical modification does not include differences onlyin nucleobase sequence.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugar moietyor a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl that is nota naturally occurring sugar moiety. Substituted sugar moieties include,but are not limited to furanosyls comprising substituents at the2′-position, the 3′-position, the 5′-position and/or the 4′-position.Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “2′-F nucleoside” refers to a nucleoside comprising asugar comprising fluorine at the 2′ position. Unless otherwiseindicated, the fluorine in a 2′-F nucleoside is in the ribo position(replacing the OH of a natural ribose).

As used herein, “2′-(ara)-F” refers to a 2′-F substituted nucleoside,wherein the fluoro group is in the arabino position.

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside sub-units are capable of linking together and/or linking toother nucleosides to form an oligomeric compound which is capable ofhybridizing to a complementary oligomeric compound. Such structuresinclude rings comprising a different number of atoms than furanosyl(e.g., 4, 6, or 7-membered rings); replacement of the oxygen of afuranosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); orboth a change in the number of atoms and a replacement of the oxygen.Such structures may also comprise substitutions corresponding to thosedescribed for substituted sugar moieties (e.g., 6-membered carbocyclicbicyclic sugar surrogates optionally comprising additionalsubstituents). Sugar surrogates also include more complex sugarreplacements (e.g., the non-ring systems of peptide nucleic acid). Sugarsurrogates include without limitation morpholinos, cyclohexenyls andcyclohexitols.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e. noadditional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “RNA-like nucleoside” means a modified nucleoside thatadopts a northern configuration and functions like RNA when incorporatedinto an oligonucleotide. RNA-like nucleosides include, but are notlimited to 3′-endo furanosyl nucleosides and RNA surrogates.

As used herein, “3′-endo-furanosyl nucleoside” means an RNA-likenucleoside that comprises a substituted sugar moiety that has a 3′-endoconformation. 3′-endo-furanosyl nucleosides include, but are not limitedto: 2′-MOE, 2′-F, 2′-OMe, LNA, ENA, and cEt nucleosides.

As used herein, “RNA-surrogate nucleoside” means an RNA-like nucleosidethat does not comprise a furanosyl. RNA-surrogate nucleosides include,but are not limited to hexitols and cyclopentanes.

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” means an atom or group of atoms bound to anoligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linking group” means any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid.

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measurable activity” means ameasurable activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenlyation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound is intended to hybridize.

As used herein, “non-target nucleic acid” means a nucleic acid moleculeto which hybridization of an antisense compound is not intended ordesired. In certain embodiments, antisense compounds do hybridize to anon-target, due to homology between the target (intended) and non-target(un-intended).

As used herein, “mRNA” means an RNA molecule that encodes a protein.

As used herein, “pre-mRNA” means an RNA transcript that has not beenfully processed into mRNA. Pre-RNA includes one or more intron.

As used herein, “object RNA” means an RNA molecule other than a targetRNA, the amount, activity, splicing, and/or function of which ismodulated, either directly or indirectly, by a target nucleic acid. Incertain embodiments, a target nucleic acid modulates splicing of anobject RNA. In certain such embodiments, an antisense compound modulatesthe amount or activity of the target nucleic acid, resulting in a changein the splicing of an object RNA and ultimately resulting in a change inthe activity or function of the object RNA.

As used herein, “microRNA” means a naturally occurring, small,non-coding RNA that represses gene expression of at least one mRNA. Incertain embodiments, a microRNA represses gene expression by binding toa target site within a 3′ untranslated region of an mRNA. In certainembodiments, a microRNA has a nucleobase sequence as set forth inmiRBase, a database of published microRNA sequences found athttp://microrna.sanger.ac.uk/sequences/. In certain embodiments, amicroRNA has a nucleobase sequence as set forth in miRBase version 12.0released September 2008, which is herein incorporated by reference inits entirety.

As used herein, “microRNA mimic” means an oligomeric compound having asequence that is at least partially identical to that of a microRNA. Incertain embodiments, a microRNA mimic comprises the microRNA seed regionof a microRNA. In certain embodiments, a microRNA mimic modulatestranslation of more than one target nucleic acids. In certainembodiments, a microRNA mimic is double-stranded.

As used herein, “differentiating nucleobase” means a nucleobase thatdiffers between two nucleic acids. In certain instances, a target regionof a target nucleic acid differs by 1-4 nucleobases from a non-targetnucleic acid. Each of those differences is referred to as adifferentiating nucleobase. In certain instances, a differentiatingnucleobase is a single-nucleotide polymorphism.

As used herein, “target-selective nucleoside” means a nucleoside of anantisense compound that corresponds to a differentiating nucleobase of atarget nucleic acid.

As used herein, “allele” means one of a pair of copies of a geneexisting at a particular locus or marker on a specific chromosome, orone member of a pair of nucleobases existing at a particular locus ormarker on a specific chromosome, or one member of a pair of nucleobasesequences existing at a particular locus or marker on a specificchromosome. For a diploid organism or cell or for autosomal chromosomes,each allelic pair will normally occupy corresponding positions (loci) ona pair of homologous chromosomes, one inherited from the mother and oneinherited from the father. If these alleles are identical, the organismor cell is said to be “homozygous” for that allele; if they differ, theorganism or cell is said to be “heterozygous” for that allele.“Wild-type allele” refers to the genotype typically not associated withdisease or dysfunction of the gene product. “Mutant allele” refers tothe genotype associated with disease or dysfunction of the gene product.

As used herein, “allelic variant” means a particular identity of anallele, where more than one identity occurs. For example, an allelicvariant may refer to either the mutant allele or the wild-type allele.

As used herein, “single nucleotide polymorphism” or “SNP” means a singlenucleotide variation between the genomes of individuals of the samespecies. In some cases, a SNP may be a single nucleotide deletion orinsertion. In general, SNPs occur relatively frequently in genomes andthus contribute to genetic diversity. The location of a SNP is generallyflanked by highly conserved sequences. An individual may be homozygousor heterozygous for an allele at each SNP site.

As used herein, “single nucleotide polymorphism site” or “SNP site”refers to the nucleotides surrounding a SNP contained in a targetnucleic acid to which an antisense compound is targeted.

As used herein, “targeting” or “targeted to” means the association of anantisense compound to a particular target nucleic acid molecule or aparticular region of a target nucleic acid molecule. An antisensecompound targets a target nucleic acid if it is sufficientlycomplementary to the target nucleic acid to allow hybridization underphysiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity under stringent conditions. Complementary oligomericcompounds need not have nucleobase complementarity at each nucleoside.Rather, some mismatches are tolerated. In certain embodiments,complementary oligomeric compounds or regions are complementary at 70%of the nucleobases (70% complementary). In certain embodiments,complementary oligomeric compounds or regions are 80% complementary. Incertain embodiments, complementary oligomeric compounds or regions are90% complementary. In certain embodiments, complementary oligomericcompounds or regions are 95% complementary. In certain embodiments,complementary oligomeric compounds or regions are 100% complementary.

As used herein, “mismatch” means a nucleobase of a first oligomericcompound that is not capable of pairing with a nucleobase at acorresponding position of a second oligomeric compound, when the firstand second oligomeric compound are aligned. Either or both of the firstand second oligomeric compounds may be oligonucleotides.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site. In certainembodiments, an antisense oligonucleotide specifically hybridizes tomore than one target site.

As used herein, “fully complementary” in reference to an oligonucleotideor portion thereof means that each nucleobase of the oligonucleotide orportion thereof is capable of pairing with a nucleobase of acomplementary nucleic acid or contiguous portion thereof. Thus, a fullycomplementary region comprises no mismatches or unhybridized nucleobasesin either strand.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “modification motif” means a pattern of chemicalmodifications in an oligomeric compound or a region thereof. Motifs maybe defined by modifications at certain nucleosides and/or at certainlinking groups of an oligomeric compound.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligomeric compound or a region thereof. Thelinkages of such an oligomeric compound may be modified or unmodified.Unless otherwise indicated, motifs herein describing only nucleosidesare intended to be nucleoside motifs. Thus, in such instances, thelinkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligomeric compound or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligomeric compound or region thereof. The nucleosides of such anoligomeric compound may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleoside have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substituent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentinvention have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms differs from the atomor a group of atoms normally present in the named functional group. Incertain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))(R_(cc))), imino(═NR_(bb)), amido (—C(O)N(R_(bb))(R_(cc)) or—N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido(—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))—R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S—(O)₂R_(bb)).Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, anoptionally linked chemical functional group or a further substituentgroup with a preferred list including without limitation, alkyl,alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,alicyclic, heterocyclic and heteroarylalkyl. Selected substituentswithin the compounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation, polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycycliccarbocyclic ring system radicals having one or more aromatic rings.Examples of aryl groups include without limitation, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ringsystems have from about 5 to about 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally include further substituentgroups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radicalcomprising a mono- or poly-cyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatoms. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroarylradicals can be attached to a parent molecule directly or through alinking moiety such as an aliphatic group or hetero atom. Heteroarylgroups as used herein may optionally include further substituent groups.

B. OLIGOMERIC COMPOUNDS

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, such oligomeric compounds compriseoligonucleotides optionally comprising one or more conjugate and/orterminal groups. In certain embodiments, an oligomeric compound consistsof an oligonucleotide. In certain embodiments, oligonucleotides compriseone or more chemical modifications. Such chemical modifications includemodifications of one or more nucleoside (including modifications to thesugar moiety and/or the nucleobase) and/or modifications to one or moreinternucleoside linkage.

a. Certain Modified Nucleosides

In certain embodiments, provided herein are oligomeric compoundscomprising or consisting of oligonucleotides comprising at least onemodified nucleoside. Such modified nucleosides comprise a modified sugarmoeity, a modified nucleobase, or both a modified sugar moiety and amodified nucleobase.

i. Certain Modified Sugar Moieties

In certain embodiments, compounds of the invention comprise one or moremodified nucleosides comprising a modified sugar moiety. Such compoundscomprising one or more sugar-modified nucleosides may have desirableproperties, such as enhanced nuclease stability or increased bindingaffinity with a target nucleic acid relative to an oligonucleotidecomprising only nucleosides comprising naturally occurring sugarmoieties. In certain embodiments, modified sugar moieties aresubstituted sugar moieties. In certain embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more non-bridging sugar substituent,including but not limited to substituents at the 2′ and/or 5′ positions.Examples of sugar substituents suitable for the 2′-position, include,but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents atthe 2′ position is selected from allyl, amino, azido, thio, O-allyl,O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examplesof sugar substituents at the 5′-position, include, but are not limitedto: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certainembodiments, substituted sugars comprise more than one non-bridgingsugar substituent, for example, 2′-F-5′-methyl sugar moieties (see,e.g., PCT International Application WO 2008/101157, for additional5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In certain embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, orN(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl,alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂—O—(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂,and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′sugar substituents, include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—; 4′-CH₂-2′,4′-(CH₂)₂-2′, 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see,e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008);4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g., WO2009/006478,published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see,e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see,e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH₂—O—N(R)-2′, and4′-CH₂—N(R)—O-2′-, wherein each R is, independently, H, a protectinggroup, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl,or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23,2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya, et al., J. Org.Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ and analogs thereof(see, published PCT International Application WO 2008/154401, publishedon Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—,—C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,(J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA, and (K) Ethylene(methoxy)(4′-(CH(CH₂OMe)-O-2′) BNA (also referred to as constrained MOE or cMOE)as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570,US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclicnucleosides have been incorporated into antisense oligonucleotides thatshowed antisense activity (Frieden et al., Nucleic Acids Research, 2003,21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCTInternational Application WO 2007/134181, published on Nov. 22, 2007,wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinylgroup).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the naturally occurringsugar is substituted, e.g., with a sulfer, carbon or nitrogen atom. Incertain such embodiments, such modified sugar moiety also comprisesbridging and/or non-bridging substituents as described above. Forexample, certain sugar surrogates comprise a 4′-sulfer atom and asubstitution at the 2′-position (see, e.g., published U.S. PatentApplication US2005/0130923, published on Jun. 16, 2005) and/or the 5′position. By way of additional example, carbocyclic bicyclic nucleosideshaving a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having otherthan 5-atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran. Such tetrahydropyrans may befurther modified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), and those compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and

each of R₁ and R₂ is independently selected from among: hydrogen,halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 Published on Aug. 21, 2008 for otherdisclosed 5′,2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, the present invention provides oligonucleotidescomprising modified nucleosides. Those modified nucleotides may includemodified sugars, modified nucleobases, and/or modified linkages. Thespecific modifications are selected such that the resultingoligonucleotides possess desirable characteristics. In certainembodiments, oligonucleotides comprise one or more RNA-like nucleosides.In certain embodiments, oligonucleotides comprise one or more DNA-likenucleotides.

ii. Certain Modified Nucleobases

In certain embodiments, nucleosides of the present invention compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present invention comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from:universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

b. Certain Internucleoside Linkages

In certain embodiments, nucleosides may be linked together using anyinternucleoside linkage to form oligonucleotides. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus containinginternucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—)thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of theoligonucleotide. In certain embodiments, internucleoside linkages havinga chiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═P)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

i. 3′-Endo Modifications

In one aspect of the present disclosure, oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base moiety, the sugar moiety or both to induce adesired 3′-endo sugar conformation. These modified nucleosides are usedto mimic RNA like nucleosides so that particular properties of anoligomeric compound can be enhanced while maintaining the desirable3′-endo conformational geometry. There is an apparent preference for anRNA type duplex (A form helix, predominantly 3′-endo) as a requirementof RNA interference which is supported in part by the fact that duplexescomposed of 2′-deoxy-2′-F-nucleosides appear efficient in triggeringRNAi response in the C. elegans system. Properties that are enhanced byusing more stable 3′-endo nucleosides include but aren't limited tomodulation of pharmacokinetic properties through modification of proteinbinding, protein off-rate, absorption and clearance; modulation ofnuclease stability as well as chemical stability; modulation of thebinding affinity and specificity of the oligomer (affinity andspecificity for enzymes as well as for complementary sequences); andincreasing efficacy of RNA cleavage. The present invention providesoligomeric compounds having one or more nucleosides modified in such away as to favor a C3′-endo type conformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element, as exemplified in Example 35, below (Gallo et al.,Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem.,(1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64,747-754.) Alternatively, preference for the 3′-endo conformation can beachieved by deletion of the 2′-OH as exemplified by 2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36,831-841), which adopts the 3′-endo conformation positioning theelectronegative fluorine atom in the axial position. Other modificationsof the ribose ring, for example substitution at the 4′-position to give4′-F modified nucleosides (Guillerm et al., Bioorganic and MedicinalChemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem.(1976), 41, 3010-3017), or for example modification to yieldmethanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett.(2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal ChemistryLetters (2001), 11, 1333-1337) also induce preference for the 3′-endoconformation. Some modifications actually lock the conformationalgeometry by formation of a bicyclic sugar moiety e.g. locked nucleicacid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylenebridged nucleic acids (ENA, Morita et al, Bioorganic & MedicinalChemistry Letters (2002), 12, 73-76.)

c. Certain Motifs

In certain embodiments, oligomeric compounds comprise or consist ofoligonucleotides. In certain embodiments, such oligonucleotides compriseone or more chemical modification. In certain embodiments, chemicallymodified oligonucleotides comprise one or more modified sugars. Incertain embodiments, chemically modified oligonucleotides comprise oneor more modified nucleobases. In certain embodiments, chemicallymodified oligonucleotides comprise one or more modified internucleosidelinkages. In certain embodiments, the chemical modifications (sugarmodifications, nucleobase modifications, and/or linkage modifications)define a pattern or motif. In certain embodiments, the patterns ofchemical modifications of sugar moieties, internucleoside linkages, andnucleobases are each independent of one another. Thus, anoligonucleotide may be described by its sugar modification motif,internucleoside linkage motif and/or nucleobase modification motif (asused herein, nucleobase modification motif describes the chemicalmodifications to the nucleobases independent of the sequence ofnucleobases).

i. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar motif. Such sugar motifs include but are not limited to any ofthe sugar modifications discussed herein.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar motif, which comprises two external regionsor “wings” and a central or internal region or “gap.” The three regionsof a gapmer sugar motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar motifs of the two wings are the same as oneanother (symmetric sugar gapmer). In certain embodiments, the sugarmotifs of the 5′-wing differs from the sugar motif of the 3′-wing(asymmetric sugar gapmer).

ii. Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certainembodiments, each nucleobase is modified. In certain embodiments, noneof the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, oligonucleotides comprise one or morenucleosides comprising a modified nucleobase. In certain embodiments,oligonucleotides having a gapmer sugar motif comprise a nucleosidecomprising a modified nucleobase. In certain such embodiments, onenucleoside comprising a modified nucleobases is in the central gap of anoligonucleotide having a gapmer sugar motif. In certain embodiments, thesugar is an unmodified 2′ deoxynucleoside. In certain embodiments, themodified nucleobase is selected from: a 2-thio pyrimidine and a5-propyne pyrimidine

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

iii. Certain Nucleoside Motifs

In certain embodiments, oligonucleotides comprise nucleosides comprisingmodified sugar moieties and/or nucleosides comprising modifiednucleobases. Such motifs can be described by their sugar motif and theirnucleobase motif separately or by their nucleoside motif, which providespositions or patterns of modified nucleosides (whether modified sugar,nucleobase, or both sugar and nucleobase) in an oligonucleotide.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer nucleoside motif, which comprises two externalregions or “wings” and a central or internal region or “gap.” The threeregions of a gapmer nucleoside motif (the 5′-wing, the gap, and the3′-wing) form a contiguous sequence of nucleosides wherein at least someof the sugar moieties and/or nucleobases of the nucleosides of each ofthe wings differ from at least some of the sugar moieties and/ornucleobase of the nucleosides of the gap. Specifically, at least thenucleosides of each wing that are closest to the gap (the 3′-mostnucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing)differ from the neighboring gap nucleosides, thus defining the boundarybetween the wings and the gap. In certain embodiments, the nucleosideswithin the gap are the same as one another. In certain embodiments, thegap includes one or more nucleoside that differs from one or more othernucleosides of the gap. In certain embodiments, the nucleoside motifs ofthe two wings are the same as one another (symmetric gapmer). In certainembodiments, the nucleoside motifs of the 5′-wing differs from thenucleoside motif of the 3′-wing (asymmetric gapmer).

iv. Certain 5′-Wings

In certain embodiments, the 5′-wing of a gapmer consists of 1 to 6linked nucleosides. In certain embodiments, the 5′-wing of a gapmerconsists of 1 to 5 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certainembodiments, the 5′-wing of a gapmer consists of 3 to 5 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of4 or 5 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 1 to 4 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 1 to 3 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 1 or 2 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of2 to 4 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 2 or 3 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 3 or 4 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 1 nucleoside.In certain embodiments, the 5′-wing of a gapmer consists of 2 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmerconsists of 4 linked nucleosides. In certain embodiments, the 5′-wing ofa gapmer consists of 5 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 6 linked nucleosides.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 5′-wing of a gapmercomprises at least two bicyclic nucleosides. In certain embodiments, the5′-wing of a gapmer comprises at least three bicyclic nucleosides. Incertain embodiments, the 5′-wing of a gapmer comprises at least fourbicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 5′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 5′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 5′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 5′-wing ofa gapmer comprises at least one 2′-substituted nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one 2′-OMe nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a non-bicyclic modified nucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is a2′-substituted nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments,each nucleoside of the 5′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the5′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 5′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 5′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer has a nucleoside motifselected from among the following: ADDA; ABDAA; ABBA; ABB; ABAA; AABAA;AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; ABADB; ABADDB; AAABB;AAAAA; ABBDC; ABDDC; ABBDCC; ABBDDC; ABBDCC; ABBC; AA; AAA; AAAA; AAAAB;AAAAAAA; AAAAAAAA; ABBB; AB; ABAB; AAAAB; AABBB; AAAAB; and AABBB,wherein each A is a modified nucleoside of a first type, each B is amodified nucleoside of a second type, each C is a modified nucleoside ofa third type, and each D is an unmodified deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer has a nucleoside motifselected from among the following: AB, ABB, AAA, BBB, BBBAA, AAB, BAA,BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB,ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA,BBBBAA, and AAABW; wherein each A is a modified nucleoside of a firsttype, each B is a modified nucleoside of a second type, and each W is amodified nucleoside of either the first type, the second type or a thirdtype.

In certain embodiments, the 5′-wing of a gapmer has a nucleoside motifselected from among the following: ABB; ABAA; AABAA; AAABAA; ABAB;ABADB; AAABB; AAAAA; AA; AAA; AAAA; AAAAB; ABBB; AB; and ABAB; whereineach A is a modified nucleoside of a first type, each B is a modifiednucleoside of a second type, and each W is a modified nucleoside ofeither the first type, the second type or a third type.

In certain embodiments, an oligonucleotide comprises any 5′-wing motifprovided herein. In certain such embodiments, the oligonucleotide is a5′-hemimer (does not comprise a 3′-wing). In certain embodiments, suchan oligonucleotide is a gapmer. In certain such embodiments, the 3′-wingof the gapmer may comprise any nucleoside motif.

In certain embodiments, the 5′-wing of a gapmer has a sugar motifselected from among those listed in the following non-limiting tables:

TABLE 1 Certain 5′-Wing Sugar Motifs Certain 5′-Wing Sugar Motifs AAAAAABCBB BABCC BCBBA CBACC AAAAB ABCBC BACAA BCBBB CBBAA AAAAC ABCCA BACABBCBBC CBBAB AAABA ABCCB BACAC BCBCA CBBAC AAABB ABCCC BACBA BCBCB CBBBAAAABC ACAAA BACBB BCBCC CBBBB AAACA ACAAB BACBC BCCAA CBBBC AAACB ACAACBACCA BCCAB CBBCA AAACC ACABA BACCB BCCAC CBBCB AABAA ACABB BACCC BCCBACBBCC AABAB ACABC BBAAA BCCBB CBCAA AABAC ACACA BBAAB BCCBC CBCAB AABBAACACB BBAAC BCCCA CBCAC AABBB ACACC BBABA BCCCB CBCBA AABBC ACBAA BBABBBCCCC CBCBB AABCA ACBAB BBABC CAAAA CBCBC AABCB ACBAC BBACA CAAAB CBCCAAABCC ACBBA BBACB CAAAC CBCCB AACAA ACBBB BBACC CAABA CBCCC AACAB ACBBCBBBAA CAABB CCAAA AACAC ACBCA BBBAB CAABC CCAAB AACBA ACBCB BBBAC CAACACCAAC AACBB ACBCC BBBBA CAACB CCABA AACBC ACCAA BBBBB CAACC CCABB AACCAACCAB BBBBC CABAA CCABC AACCB ACCAC BBBCA CABAB CCACA AACCC ACCBA BBBCBCABAC CCACB ABAAA ACCBB BBBCC CABBA CCACC ABAAB ACCBC BBCAA CABBB CCBAAABAAC ACCCA BBCAB CABBC CCBAB ABABA ACCCB BBCAC CABCA CCBAC ABABB ACCCCBBCBA CABCB CCBBA ABABC BAAAA BBCBB CABCC CCBBB ABACA BAAAB BBCBC CACAACCBBC ABACB BAAAC BBCCA CACAB CCBCA ABACC BAABA BBCCB CACAC CCBCB ABBAABAABB BBCCC CACBA CCBCC ABBAB BAABC BCAAA CACBB CCCAA ABBAC BAACA BCAABCACBC CCCAB ABBBA BAACB BCAAC CACCA CCCAC ABBBB BAACC BCABA CACCB CCCBAABBBC BABAA BCABB CACCC CCCBB ABBCA BABAB BCABC CBAAA CCCBC ABBCB BABACBCACA CBAAB CCCCA ABBCC BABBA BCACB CBAAC CCCCB ABCAA BABBB BCACC CBABACCCCC ABCAB BABBC BCBAA CBABB ABCAC BABCA BCBAB CBABC ABCBA BABCB BCBACCBACA

TABLE 2 Certain 5′-Wing Sugar Motifs Certain 5′-Wing Sugar Motifs AAAAABABC CBAB ABBB BAA AAAAB BACA CBAC BAAA BAB AAABA BACB CBBA BAAB BBAAAABB BACC CBBB BABA BBB AABAA BBAA CBBC BABB AA AABAB BBAB CBCA BBAA ABAABBA BBAC CBCB BBAB AC AABBB BBBA CBCC BBBA BA ABAAA BBBB CCAA BBBB BBABAAB BBBC CCAB AAA BC ABABA BBCA CCAC AAB CA ABABB BBCB CCBA AAC CBABBAA BBCC CCBB ABA CC ABBAB BCAA CCBC ABB AA ABBBA BCAB CCCA ABC ABABBBB BCAC CCCB ACA BA BAAAA ABCB BCBA ACB BAAAB ABCC BCBB ACC BAABAACAA BCBC BAA BAABB ACAB BCCA BAB BABAA ACAC BCCB BAC BABAB ACBA BCCCBBA BABBA ACBB CAAA BBB BABBB ACBC CAAB BBC BBAAA ACCA CAAC BCA BBAABACCB CABA BCB BBABA ACCC CABB BCC BBABB BAAA CABC CAA BBBAA BAAB CACACAB BBBAB BAAC CACB CAC BBBBA BABA CACC CBA BBBBB BABB CBAA CBB AAAAAACC CCCC CBC AAAB ABAA AAAA CCA AAAC ABAB AAAB CCB AABA ABAC AABA CCCAABB ABBA AABB AAA AABC ABBB ABAA AAB AACA ABBC ABAB ABA AACB ABCA ABBAABB

In certain embodiments, each A, each B, and each C located at the3′-most 5′-wing nucleoside is a modified nucleoside. For example, incertain embodiments the 5′-wing motif is selected from among ABB, BBB,and CBB, wherein the underlined nucleoside represents the 3′-most5′-wing nucleoside and wherein the underlined nucleoside is a modifiednucleoside. In certain embodiments, the 3′-most 5′-wing nucleosidecomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, the 3′-most5′-wing nucleoside comprises a bicyclic sugar moiety selected from amongcEt and LNA. In certain embodiments, the 3′-most 5′-wing nucleosidecomprises cEt. In certain embodiments, the 3′-most 5′-wing nucleosidecomprises LNA.

In certain embodiments, each A comprises an unmodified 2′-deoxyfuranosesugar moiety. In certain embodiments, each A comprises a modified sugarmoiety. In certain embodiments, each A comprises a 2′-substituted sugarmoiety. In certain embodiments, each A comprises a 2′-substituted sugarmoiety selected from among F, ara-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each A comprises a bicyclic sugar moiety. In certainembodiments, each A comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each A comprises a modified nucleobase. In certainembodiments, each A comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certainembodiments, each A comprises an HNA. In certain embodiments, each Acomprises a F-HNA. In certain embodiments, each A comprises a5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-MeDNA.

In certain embodiments, each B comprises an unmodified 2′-deoxyfuranosesugar moiety. In certain embodiments, each B comprises a modified sugarmoiety. In certain embodiments, each B comprises a 2′-substituted sugarmoiety. In certain embodiments, each B comprises a 2′-substituted sugarmoiety selected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each B comprises a bicyclic sugar moiety. In certainembodiments, each B comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each B comprises a modified nucleobase. In certainembodiments, each B comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. Incertain embodiments, each B comprises an HNA. In certain embodiments,each B comprises a F-HNA. In certain embodiments, each B comprises a5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-MeDNA.

In certain embodiments, each A comprises a 2′-substituted sugar moietyselected from among F, ara-F, OCH₃ and O(CH₂)₂—OCH₃ and each B comprisesa bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENAand 2′-thio LNA. In certain embodiments, each A comprises O(CH₂)₂—OCH₃and each B comprises cEt.

In certain embodiments, each C comprises an unmodified 2′-deoxyfuranosesugar moiety. In certain embodiments, each C comprises a modified sugarmoiety. In certain embodiments, each C comprises a 2′-substituted sugarmoiety. In certain embodiments, each C comprises a 2′-substituted sugarmoiety selected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each C comprises a 5′-substituted sugar moiety. In certainembodiments, each C comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me DNA, and 5′-(R)-Me DNA. In certain embodiments, each Ccomprises a bicyclic sugar moiety. In certain embodiments, each Ccomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises amodified nucleobase. In certain embodiments, each C comprises a modifiednucleobase selected from among 2-thio-thymidine and 5-propyne uridine.In certain embodiments, each C comprises a 2-thio-thymidine nucleoside.In certain embodiments, each C comprises an HNA. In certain embodiments,each C comprises an F-HNA.

v. Certain 3′-Wings

In certain embodiments, the 3′-wing of a gapmer consists of 1 to 6linked nucleosides. In certain embodiments, the 3′-wing of a gapmerconsists of 1 to 5 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certainembodiments, the 3′-wing of a gapmer consists of 3 to 5 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of4 or 5 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 1 to 4 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 1 to 3 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 1 or 2 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of2 to 4 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 2 or 3 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 3 or 4 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 1 nucleoside.In certain embodiments, the 3′-wing of a gapmer consists of 2 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of31 inked nucleosides. In certain embodiments, the 3′-wing of a gapmerconsists of 4 linked nucleosides. In certain embodiments, the 3′-wing ofa gapmer consists of 5 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 6 linked nucleosides.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 3′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 3′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 3′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least two non-bicyclic modified nucleosides. Incertain embodiments, the 3′-wing of a gapmer comprises at least threenon-bicyclic modified nucleosides. In certain embodiments, the 3′-wingof a gapmer comprises at least four non-bicyclic modified nucleosides.In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-substituted nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one 2′-OMe nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is anon-bicyclic modified nucleoside. In certain embodiments, eachnucleoside of the 3′-wing of a gapmer is a 2′-substituted nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is a2′-MOE nucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 3′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneLNA nucleoside and at least one non-bicyclic modified nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-substituted nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside and atleast one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one LNA nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one non-bicyclic modified nucleoside, andat least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least one constrained ethyl nucleoside, at leastone non-bicyclic modified nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one LNA nucleoside, at least one non-bicyclicmodified nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-substituted nucleoside, and atleast one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one constrained ethyl nucleoside, at least one2′-substituted nucleoside, and at least one 2′-deoxynucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside, at least one 2′-substituted nucleoside, and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-MOEnucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-OMenucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer has a nucleoside motifselected from among the following: ABB, ABAA, AAABAA, AAAAABAA, AABAA,AAAABAA, AAABAA, ABAB, AAAAA, AAABB, AAAAAAAA, AAAAAAA, AAAAAA, AAAAB,AAAA, AAA, AA, AB, ABBB, ABAB, AABBB; wherein each A is a modifiednucleoside of a first type, each B is a modified nucleoside of a secondtype. In certain embodiments, an oligonucleotide comprises any 3′-wingmotif provided herein. In certain such embodiments, the oligonucleotideis a 3′-hemimer (does not comprise a 5′-wing). In certain embodiments,such an oligonucleotide is a gapmer. In certain such embodiments, the5′-wing of the gapmer may comprise any nucleoside motif.

In certain embodiments, the 3′-wing of a gapmer has a nucleoside motifselected from among the following: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA,WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA,BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB, wherein each A isa modified nucleoside of a first type, each B is a modified nucleosideof a second type, and each W is a modified nucleoside of either thefirst type, the second type or a third type.

In certain embodiments, the 3′-wing of a gapmer has a nucleoside motifselected from among the following: ABB; AAABAA; AABAA; AAAABAA; AAAAA;AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AB; ABBB; and ABAB, whereineach A is a modified nucleoside of a first type, each B is a modifiednucleoside of a second type, and each W is a modified nucleoside ofeither the first type, the second type or a third type.

In certain embodiments, the 3′-wing of a gapmer has a sugar motifselected from among those listed in the following non-limiting tables:

TABLE 3 Certain 3′-Wing Sugar Motifs Certain 3′-Wing Sugar Motifs AAAAAABCBB BABCC BCBBA CBACC AAAAB ABCBC BACAA BCBBB CBBAA AAAAC ABCCA BACABBCBBC CBBAB AAABA ABCCB BACAC BCBCA CBBAC AAABB ABCCC BACBA BCBCB CBBBAAAABC ACAAA BACBB BCBCC CBBBB AAACA ACAAB BACBC BCCAA CBBBC AAACB ACAACBACCA BCCAB CBBCA AAACC ACABA BACCB BCCAC CBBCB AABAA ACABB BACCC BCCBACBBCC AABAB ACABC BBAAA BCCBB CBCAA AABAC ACACA BBAAB BCCBC CBCAB AABBAACACB BBAAC BCCCA CBCAC AABBB ACACC BBABA BCCCB CBCBA AABBC ACBAA BBABBBCCCC CBCBB AABCA ACBAB BBABC CAAAA CBCBC AABCB ACBAC BBACA CAAAB CBCCAAABCC ACBBA BBACB CAAAC CBCCB AACAA ACBBB BBACC CAABA CBCCC AACAB ACBBCBBBAA CAABB CCAAA AACAC ACBCA BBBAB CAABC CCAAB AACBA ACBCB BBBAC CAACACCAAC AACBB ACBCC BBBBA CAACB CCABA AACBC ACCAA BBBBB CAACC CCABB AACCAACCAB BBBBC CABAA CCABC AACCB ACCAC BBBCA CABAB CCACA AACCC ACCBA BBBCBCABAC CCACB ABAAA ACCBB BBBCC CABBA CCACC ABAAB ACCBC BBCAA CABBB CCBAAABAAC ACCCA BBCAB CABBC CCBAB ABABA ACCCB BBCAC CABCA CCBAC ABABB ACCCCBBCBA CABCB CCBBA ABABC BAAAA BBCBB CABCC CCBBB ABACA BAAAB BBCBC CACAACCBBC ABACB BAAAC BBCCA CACAB CCBCA ABACC BAABA BBCCB CACAC CCBCB ABBAABAABB BBCCC CACBA CCBCC ABBAB BAABC BCAAA CACBB CCCAA ABBAC BAACA BCAABCACBC CCCAB ABBBA BAACB BCAAC CACCA CCCAC ABBBB BAACC BCABA CACCB CCCBAABBBC BABAA BCABB CACCC CCCBB ABBCA BABAB BCABC CBAAA CCCBC ABBCB BABACBCACA CBAAB CCCCA ABBCC BABBA BCACB CBAAC CCCCB ABCAA BABBB BCACC CBABACCCCC ABCAB BABBC BCBAA CBABB ABCAC BABCA BCBAB CBABC ABCBA BABCB BCBACCBACA

TABLE 4 Certain 3′-Wing Sugar Motifs Certain 3′-Wing Sugar Motifs AAAAABABC CBAB ABBB BAA AAAAB BACA CBAC BAAA BAB AAABA BACB CBBA BAAB BBAAAABB BACC CBBB BABA BBB AABAA BBAA CBBC BABB AA AABAB BBAB CBCA BBAA ABAABBA BBAC CBCB BBAB AC AABBB BBBA CBCC BBBA BA ABAAA BBBB CCAA BBBB BBABAAB BBBC CCAB  AAA BC ABABA BBCA CCAC  AAB CA ABABB BBCB CCBA  AAC CBABBAA BBCC CCBB  ABA CC ABBAB BCAA CCBC  ABB AA ABBBA BCAB CCCA  ABC ABABBBB BCAC CCCB  ACA BA BAAAA ABCB BCBA ACB BAAAB ABCC BCBB ACC BAABAACAA BCBC BAA BAABB ACAB BCCA BAB BABAA ACAC BCCB BAC BABAB ACBA BCCCBBA BABBA ACBB CAAA BBB BABBB ACBC CAAB BBC BBAAA ACCA CAAC BCA BBAABACCB CABA BCB BBABA ACCC CABB BCC BBABB BAAA CABC CAA BBBAA BAAB CACACAB BBBAB BAAC CACB CAC BBBBA BABA CACC CBA BBBBB BABB CBAA CBB AAAAAACC CCCC CBC AAAB ABAA AAAA CCA AAAC ABAB AAAB CCB AABA ABAC AABA CCCAABB ABBA AABB AAA AABC ABBB ABAA AAB AACA ABBC ABAB ABA AACB ABCA ABBAABB

In certain embodiments, each A, each B, and each C located at the5′-most 3′-wing region nucleoside is a modified nucleoside. For example,in certain embodiments the 3′-wing motif is selected from among ABB,BBB, and CBB, wherein the underlined nucleoside represents the 5′-most3′-wing region nucleoside and wherein the underlined nucleoside is amodified nucleoside.

In certain embodiments, each A comprises an unmodified 2′-deoxyfuranosesugar moiety. In certain embodiments, each A comprises a modified sugarmoiety. In certain embodiments, each A comprises a 2′-substituted sugarmoiety. In certain embodiments, each A comprises a 2′-substituted sugarmoiety selected from among F, ara-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each A comprises a bicyclic sugar moiety. In certainembodiments, each A comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each A comprises a modified nucleobase. In certainembodiments, each A comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certainembodiments, each A comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me DNA, and 5′-(R)-Me DNA.

In certain embodiments, each B comprises an unmodified 2′-deoxyfuranosesugar moiety. In certain embodiments, each B comprises a modified sugarmoiety. In certain embodiments, each B comprises a 2′-substituted sugarmoiety. In certain embodiments, each B comprises a 2′-substituted sugarmoiety selected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each B comprises a bicyclic sugar moiety. In certainembodiments, each B comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each B comprises a modified nucleobase. In certainembodiments, each B comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. Incertain embodiments, each B comprises an HNA. In certain embodiments,each B comprises an F-HNA. In certain embodiments, each B comprises a5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-MeDNA.

In certain embodiments, each A comprises a 2′-substituted sugar moietyselected from among F, ara-F, OCH₃ and O(CH₂)₂—OCH₃ and each B comprisesa bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENAand 2′-thio LNA. In certain embodiments, each A comprises O(CH₂)₂—OCH₃and each B comprises cEt.

In certain embodiments, each C comprises an unmodified 2′-deoxyfuranosesugar moiety. In certain embodiments, each C comprises a modified sugarmoiety. In certain embodiments, each C comprises a 2′-substituted sugarmoiety. In certain embodiments, each C comprises a 2′-substituted sugarmoiety selected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each C comprises a 5′-substituted sugar moiety. In certainembodiments, each C comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me, and 5′-(R)-Me. In certain embodiments, each Ccomprises a bicyclic sugar moiety. In certain embodiments, each Ccomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises amodified nucleobase. In certain embodiments, each C comprises a modifiednucleobase selected from among 2-thio-thymidine and 5-propyne uridine.In certain embodiments, each C comprises a 2-thio-thymidine nucleoside.In certain embodiments, each C comprises an HNA. In certain embodiments,each C comprises an F-HNA.

vi. Certain Central Regions (Gaps)

In certain embodiments, the gap of a gapmer consists of 6 to 20 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6to 15 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 to 12 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 6 to 10 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 6 to 8 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6or 7 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 7 to 10 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 7 to 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 8 to 10 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 8or 9 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 linked nucleosides. In certain embodiments, the gap of agapmer consists of 7 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 8 linked nucleosides. In certain embodiments,the gap of a gapmer consists of 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 10 linked nucleosides. Incertain embodiments, the gap of a gapmer consists of 11 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 12linked nucleosides.

In certain embodiments, each nucleoside of the gap of a gapmer is a2′-deoxynucleoside. In certain embodiments, the gap comprises one ormore modified nucleosides. In certain embodiments, each nucleoside ofthe gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleosidethat is “DNA-like.” In such embodiments, “DNA-like” means that thenucleoside has similar characteristics to DNA, such that a duplexcomprising the gapmer and an RNA molecule is capable of activating RNaseH. For example, under certain conditions, 2′-(ara)-F have been shown tosupport RNase H activation, and thus is DNA-like. In certainembodiments, one or more nucleosides of the gap of a gapmer is not a2′-deoxynucleoside and is not DNA-like. In certain such embodiments, thegapmer nonetheless supports RNase H activation (e.g., by virtue of thenumber or placement of the non-DNA nucleosides).

In certain embodiments, gaps comprise a stretch of unmodified2′-deoxynucleoside interrupted by one or more modified nucleosides, thusresulting in three sub-regions (two stretches of one or more2′-deoxynucleosides and a stretch of one or more interrupting modifiednucleosides). In certain embodiments, no stretch of unmodified2′-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certainembodiments, such short stretches is achieved by using short gapregions. In certain embodiments, short stretches are achieved byinterrupting a longer gap region.

In certain embodiments, the gap comprises one or more modifiednucleosides. In certain embodiments, the gap comprises one or moremodified nucleosides selected from among cEt, FHNA, LNA, and2-thio-thymidine. In certain embodiments, the gap comprises one modifiednucleoside. In certain embodiments, the gap comprises a 5′-substitutedsugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certainembodiments, the gap comprises two modified nucleosides. In certainembodiments, the gap comprises three modified nucleosides. In certainembodiments, the gap comprises four modified nucleosides. In certainembodiments, the gap comprises two or more modified nucleosides and eachmodified nucleoside is the same. In certain embodiments, the gapcomprises two or more modified nucleosides and each modified nucleosideis different.

In certain embodiments, the gap comprises one or more modified linkages.In certain embodiments, the gap comprises one or more methyl phosphonatelinkages. In certain embodiments the gap comprises two or more modifiedlinkages. In certain embodiments, the gap comprises one or more modifiedlinkages and one or more modified nucleosides. In certain embodiments,the gap comprises one modified linkage and one modified nucleoside. Incertain embodiments, the gap comprises two modified linkages and two ormore modified nucleosides.

In certain embodiments, the gap comprises a nucleoside motif selectedfrom among the following: DDDDXDDDDD; DDDDDXDDDDD; DDDXDDDDD;DDDDXDDDDDD; DDDDXDDDD; DDXDDDDDD; DDDXDDDDDD; DXDDDDDD; DDXDDDDDDD;DDXDDDDD; DDXDDDXDDD; DDDXDDDXDDD; DXDDDXDDD; DDXDDDXDD; DDXDDDDXDDD;DDXDDDDXDD; DXDDDDXDDD; DDDDXDDD; DDDXDDD; DXDDDDDDD; DDDDXXDDD; andDXXDXXDXX; wherein each D is an unmodified deoxynucleoside; and each Xis a modified nucleoside or a substituted sugar moiety.

In certain embodiments, the gap comprises a nucleoside motif selectedfrom among the following: DDDDDDDDD; DXDDDDDDD; DDXDDDDDD; DDDXDDDDD;DDDDXDDDD; DDDDDXDDD; DDDDDDXDD; DDDDDDDXD; DXXDDDDDD; DDDDDDXXD;DDXXDDDDD; DDDXXDDDD; DDDDXXDDD; DDDDDXXDD; DXDDDDDXD; DXDDDDXDD;DXDDDXDDD; DXDDXDDDD; DXDXDDDDD; DDXDDDDXD; DDXDDDXDD; DDXDDXDDD;DDXDXDDDD; DDDXDDDXD; DDDXDDXDD; DDDXDXDDD; DDDDXDDXD; DDDDXDXDD; andDDDDDXDXD, wherein each D is an unmodified deoxynucleoside; and each Xis a modified nucleoside or a substituted sugar moiety.

In certain embodiments, the gap comprises a nucleoside motif selectedfrom among the following: DDDDXDDDD, DXDDDDDDD, DXXDDDDDD, DDXDDDDDD,DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, and DDDDDDDXD, wherein eachD is an unmodified deoxynucleoside; and each X is a modified nucleosideor a substituted sugar moiety.

In certain embodiments, the gap comprises a nucleoside motif selectedfrom among the following: DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD,DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD,DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD,DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD, wherein each D is anunmodified deoxynucleoside; and each X is a modified nucleoside or asubstituted sugar moiety.

In certain embodiments, the gap comprises a nucleoside motif selectedfrom among the following: DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD,DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD,DDDXDXD, and DDDDXXD, wherein each D is an unmodified deoxynucleoside;and each X is a modified nucleoside or a substituted sugar moiety.

In certain embodiments, the gap comprises a nucleoside motif selectedfrom among the following: DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD,DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD, wherein each D is anunmodified deoxynucleoside; and each X is a modified nucleoside or asubstituted sugar moiety.

In certain embodiments, the gap comprises a nucleoside motif selectedfrom among the following: DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXDDDDD,DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDDDD, DDXDDDDD, DDDXDDDD,DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDDDD; DDXDDDDDD, DDDXDDDDD,DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXDDDDDDDD, DDXDDDDDDD,DDDXDDDDDD, DDDDXDDDDD, DDDDDXDDDD, DDDDDDXDDD, DDDDDDDXDD, andDDDDDDDDXD, wherein each D is an unmodified deoxynucleoside; and each Xis a modified nucleoside or a substituted sugar moiety.

In certain embodiments, each X comprises an unmodified 2′-deoxyfuranosesugar moiety. In certain embodiments, each X comprises a modified sugarmoiety. In certain embodiments, each X comprises a 2′-substituted sugarmoiety. In certain embodiments, each X comprises a 2′-substituted sugarmoiety selected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each X comprises a 5′-substituted sugar moiety. In certainembodiments, each X comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me, and 5′-(R)-Me. In certain embodiments, each Xcomprises a bicyclic sugar moiety. In certain embodiments, each Xcomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each X comprises amodified nucleobase. In certain embodiments, each X comprises a modifiednucleobase selected from among 2-thio-thymidine and 5-propyne uridine.In certain embodiments, each X comprises a 2-thio-thymidine nucleoside.In certain embodiments, each X comprises an HNA. In certain embodiments,each C comprises an F-HNA. In certain embodiments, X represents thelocation of a single differentiating nucleobase.

vii. Certain Gapmer Motifs

In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′wing, wherein the 5′-wing, gap, and 3′ wing are independently selectedfrom among those discussed above. For example, in certain embodiments, agapmer has a 5′-wing, a gap, and a 3′-wing having features selected fromamong any of those listed in the tables above and any 5′-wing may bepaired with any gap and any 3′-wing. For example, in certainembodiments, a 5′-wing may comprise AAABB, a 3′-wing may comprise BBA,and the gap may comprise DDDDDDD. For example, in certain embodiments, agapmer has a 5′-wing, a gap, and a 3′-wing having features selected fromamong those listed in the following non-limiting table, wherein eachmotif is represented as (5′-wing)-(gap)-(3′-wing), wherein each numberrepresents the number of linked nucleosides in each portion of themotif, for example, a 5-10-5 motif would have a 5′-wing comprising 5nucleosides, a gap comprising 10 nucleosides, and a 3′-wing comprising 5nucleosides:

TABLE 5 Certain Gapmer Sugar Motifs Certain Gapmer Sugar Motifs 2-10-23-10-2 4-10-2 5-10-2 2-10-3 3-10-3 4-10-3 5-10-3 2-10-4 3-10-4 4-10-45-10-4 2-10-5 3-10-5 4-10-5 5-10-5 2-9-2 3-9-2 4-9-2 5-9-2 2-9-3 3-9-34-9-3 5-9-3 2-9-4 3-9-4 4-9-4 5-9-4 2-9-5 3-9-5 4-9-5 5-9-5 2-11-23-11-2 4-11-2 5-11-2 2-11-3 3-11-3 4-11-3 5-11-3 2-11-4 3-11-4 4-11-45-11-4 2-11-5 3-11-5 4-11-5 5-11-5 2-8-2 3-8-2 4-8-2 5-8-2 2-8-3 3-8-34-8-3 5-8-3 2-8-4 3-8-4 4-8-4 5-8-4 2-8-5 3-8-5 4-8-5 5-8-5

In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′wing, wherein the 5′-wing, gap, and 3′ wing are independently selectedfrom among those discussed above. For example, in certain embodiments, agapmer has a 5′-wing, a gap, and a 3′-wing having features selected fromamong those listed in the following non-limiting tables:

TABLE 6 Certain Gapmer Nucleoside Motifs 5′-wing Central gap 3′-wingregion region region ADDA DDDDDD ABB ABBA DDDADDDD ABAA AAAAAAADDDDDDDDDDD AAA AAAAABB DDDDDDDD BBAAAAA ABB DDDDADDDD ABB ABB DDDDBDDDDBBA ABB DDDDDDDDD BBA AABAA DDDDDDDDD AABAA ABB DDDDDD AABAA AAABAADDDDDDDDD AAABAA AAABAA DDDDDDDDD AAB ABAB DDDDDDDDD ABAB AAABB DDDDDDDBBA ABADB DDDDDDD BBA ABA DBDDDDDDD BBA ABA DADDDDDDD BBA ABAB DDDDDDDDBBA AA DDDDDDDD BBBBBBBB ABB DDDDDD ABADB AAAAB DDDDDDD BAAAA ABBBDDDDDDDDD AB AB DDDDDDDDD BBBA ABBB DDDDDDDDD BBBA AB DDDDDDDD ABA ABBDDDDWDDDD BBA AAABB DDDWDDD BBAAA ABB DDDDWWDDD BBA ABADB DDDDDDD BBAABBDC DDDDDDD BBA ABBDDC DDDDDD BBA ABBDCC DDDDDD BBA ABB DWWDWWDWW BBAABB DWDDDDDDD BBA ABB DDWDDDDDD BBA ABB DWWDDDDDD BBA AAABB DDWDDDDDD AABB DDWDWDDDD BBABBBB ABB DDDD(^(N)D)DDDD BBA AAABB DDD(^(N)D)DDD BBAAAABB DDDD(^(N)D)(^(N)D)DDD BBA ABBD(^(N)D)(^(N)D)D(^(N)D)(^(N)D)D(^(N)D)(^(N)D) BBA ABB D(^(N)D)DDDDDDDBBA ABB DD(^(N)D)DDDDDD BBA ABB D(^(N)D)(^(N)D)DDDDDD BBA AAABBDD(^(N)D)DDDDDD AA BB DD(^(N)D)D(^(N)D)DDDD BBABBBB ABAB DDDDDDDDD BABA

TABLE 7 Certain Gapmer Nucleoside Motifs 5′-wing Central gap 3′-wingregion region region ABBW DDDDDDDD BBA ABB DWDDDDDDD BBA ABB DDWDDDDDDBBA ABB DDDWDDDDD BBA ABB DDDDWDDDD BBA ABB DDDDDWDDD BBA ABB DDDDDDWDDBBA ABB DDDDDDDWD BBA ABB DDDDDDDD WBBA ABBWW DDDDDDD BBA ABB DWWDDDDDDBBA ABB DDWWDDDDD BBA ABB DDDWWDDDD BBA ABB DDDDWWDDD BBA ABB DDDDDWWDDBBA ABB DDDDDDWWD BBA ABB DDDDDDD WWBBA ABBW DDDDDDD WBBA ABBW DDDDDDWDBBA ABBW DDDDDWDD BBA ABBW DDDDWDDD BBA ABBW DDDWDDDD BBA ABBW DDWDDDDDBBA ABBW DWDDDDDD BBA ABB DWDDDDDD WBBA ABB DWDDDDDWD BBA ABB DWDDDDWDDBBA ABB DWDDDWDDD BBA ABB DWDDWDDDD BBA ABB DWDWDDDDD BBA ABB DDWDDDDDWBBA ABB DDWDDDDWD BBA ABB DDWDDDWDD BBA ABB DDWDDWDDD BBA ABB DDWDWDDDDBBA ABB DDWWDDDDD BBA ABB DDDWDDDD WBBA ABB DDDWDDDWD BBA ABB DDDWDDWDDBBA ABB DDDWDWDDD BBA ABB DDDWWDDDD BBA ABB DDDDWDDD WBBA ABB DDDDWDDWDBBA ABB DDDDWDWDD BBA ABB DDDDWWDDD BBA ABB DDDDDWDD WBBA ABB DDDDDWDWDBBA ABB DDDDDWWDD BBA ABB DDDDDDWD WBBA

TABLE 8 Certain Gapmer Nucleoside Motifs 5′-wing Central gap 3′-wingregion region region ABBB DDDDDDDD BBA ABB DBDDDDDDD BBA ABB DDBDDDDDDBBA ABB DDDBDDDDD BBA ABB DDDDBDDDD BBA ABB DDDDDBDDD BBA ABB DDDDDDBDDBBA ABB DDDDDDDBD BBA ABB DDDDDDDD BBBA ABBBB DDDDDDD BBA ABB DBBDDDDDDBBA ABB DDBBDDDDD BBA ABB DDDBBDDDD BBA ABB DDDDBBDDD BBA ABB DDDDDBBDDBBA ABB DDDDDDBBD BBA ABB DDDDDDD BBBBA ABBB DDDDDDD BBBA ABB DDDDDDBDBBA ABBB DDDDDBDD BBA ABBB DDDDBDDD BBA ABBB DDDBDDDD BBA ABBB DDBDDDDDBBA ABBB DBDDDDDD BBA ABB DBDDDDDD BBBA ABB DBDDDDDBD BBA ABB DBDDDDBDDBBA ABB DBDDDBDDD BBA ABB DBDDBDDDD BBA ABB DBDBDDDDD BBA ABB DDBDDDDDBBBA ABB DDBDDDDBD BBA ABB DDBDDDBDD BBA ABB DDBDDBDDD BBA ABB DDBDBDDDDBBA ABB DDBBDDDDD BBA ABB DDDBDDDD BBBA ABB DDDBDDDBD BBA ABB DDDBDDBDDBBA ABB DDDBDBDDD BBA ABB DDDBBDDDD BBA ABB DDDDBDDD BBBA ABB DDDDBDDBDBBA ABB DDDDBDBDD BBA ABB DDDDBBDDD BBA ABB DDDDDBDD BBBA ABB DDDDDBDBDBBA ABB DDDDDBBDD BBA ABB DDDDDDBD BBBA

TABLE 9 Certain Gapmer Nucleoside Motifs 5′-wing Central gap 3′-wingregion region region ABB DDDDDDDDD BBA AB DBDDDDDDDD BBA AB DDBDDDDDDDBBA AB DDDBDDDDDD BBA AB DDDDBDDDDD BBA AB DDDDDBDDDD BBA AB DDDDDDBDDDBBA AB DDDDDDDBDD BBA AB DDDDDDDDBD BBA AB DDDDDDDDD BBBA ABBB DDDDDDDDBBA AB DBBDDDDDDD BBA AB DDBBDDDDDD BBA AB DDDBBDDDDD BBA AB DDDDBBDDDDBBA AB DDDDDBBDDD BBA AB DDDDDDBBDD BBA AB DDDDDDDBBD BBA AB DDDDDDDDBBBBA ABBBB DDDDDDD BBA AB DBBBDDDDDD BBA AB DDBBBDDDDD BBA ABDDDBBBDDDD BBA AB DDDDBBBDDD BBA AB DDDDDBBBDD BBA AB DDDDDDBBBD BBA ABDDDDDDD BBBBBA AB DDDDDDDDD BBBA AB DDDDDDDBD BBBA AB DDDDDBDD BBBA ABDDDDBDDD BBBA AB DDDBDDDD BBBA AB DDBDDDDD BBBA AB DBDDDDDD BBBA ABDDDDDBD BBBBA AB DDDDBDD BBBBA AB DDDBDDD BBBBA AB DDBDDDD BBBBA ABDBDDDDD BBBBA AB DDDDBD BBBBBA AB DDDBDD BBBBBA AB DDBDDD BBBBBA ABDBDDDD BBBBBA

TABLE 10 Certain Gapmer Nucleoside Motifs 5′-wing Central gap 3′-wingregion region region AAAAAA DDDDDDD BABA AAAAAB DDDDDDD BABA AAAABADDDDDDD BABA AAABAA DDDDDDD BABA AABAAA DDDDDDD BABA ABAAAA DDDDDDD BABABAAAAA DDDDDDD BABA ABAAAB DDDDDDD BABA ABAABA DDDDDDD BABA ABABAADDDDDDD BABA ABBAAA DDDDDDD BABA AABAAB DDDDDDD BABA AABABA DDDDDDD BABAAABBAA DDDDDDD BABA AAABAB DDDDDDD BABA AAABBA DDDDDDD BABA AAAABBDDDDDDD BABA BAAAAB DDDDDDD BABA BAAABA DDDDDDD BABA BAABAA DDDDDDD BABABABAAA DDDDDDD BABA BBAAAA DDDDDDD BABA BBBAAA DDDDDDD BABA BBABAADDDDDDD BABA BBAABA DDDDDDD BABA BBAAAB DDDDDDD BABA ABABAB DDDDDDD BABABBBBAA DDDDDDD BABA BBBABA DDDDDDD BABA BBBAAB DDDDDDD BABA BBBBBADDDDDDD BABA BBBBAB DDDDDDD BABA AAABBB DDDDDDD BABA AABABB DDDDDDD BABAABAABB DDDDDDD BABA BAAABB DDDDDDD BABA AABBBB DDDDDDD BABA ABABBBDDDDDDD BABA BAABBB DDDDDDD BABA ABBBBB DDDDDDD BABA BABBBB DDDDDDD BABABBBBBB DDDDDDD BABA

TABLE 11 Certain Gapmer Nucleoside Motifs 5′-wing Central gap 3′-wingregion region region AAAAA DDDDDDD AAAAA AAAAB DDDDDDD AAAAA AAABADDDDDDD AAAAA AAABB DDDDDDD AAAAA AABAA DDDDDDD AAAAA AABAB DDDDDDDAAAAA AABBA DDDDDDD AAAAA AABBB DDDDDDD AAAAA ABAAA DDDDDDD AAAAA ABAABDDDDDDD AAAAA ABABA DDDDDDD AAAAA ABABB DDDDDDD AAAAA ABBAA DDDDDDDAAAAA ABBAB DDDDDDD AAAAA ABBBA DDDDDDD AAAAA ABBBB DDDDDDD AAAAA BAAAADDDDDDD AAAAA BAAAB DDDDDDD AAAAA BAABA DDDDDDD AAAAA BAABB DDDDDDDAAAAA BABAA DDDDDDD AAAAA BABAB DDDDDDD AAAAA BABBA DDDDDDD AAAAA BABBBDDDDDDD AAAAA BBAAA DDDDDDD AAAAA BBAAB DDDDDDD AAAAA BBABA DDDDDDDAAAAA BBABB DDDDDDD AAAAA BBBAA DDDDDDD AAAAA BBBAB DDDDDDD AAAAA BBBBADDDDDDD AAAAA BBBBB DDDDDDD AAAAA AAAAA DDDDDDD BAAAA AAAAB DDDDDDDBAAAA AAABA DDDDDDD BAAAA AAABB DDDDDDD BAAAA AABAA DDDDDDD BAAAA AABABDDDDDDD BAAAA AABBA DDDDDDD BAAAA AABBB DDDDDDD BAAAA ABAAA DDDDDDDBAAAA ABAAB DDDDDDD BAAAA ABABA DDDDDDD BAAAA ABABB DDDDDDD BAAAA ABBAADDDDDDD BAAAA ABBAB DDDDDDD BAAAA ABBBA DDDDDDD BAAAA ABBBB DDDDDDDBAAAA BAAAA DDDDDDD BAAAA BAAAB DDDDDDD BAAAA BAABA DDDDDDD BAAAA BAABBDDDDDDD BAAAA BABAA DDDDDDD BAAAA BABAB DDDDDDD BAAAA BABBA DDDDDDDBAAAA BABBB DDDDDDD BAAAA BBAAA DDDDDDD BAAAA BBAAB DDDDDDD BAAAA BBABADDDDDDD BAAAA BBABB DDDDDDD BAAAA BBBAA DDDDDDD BAAAA BBBAB DDDDDDDBAAAA BBBBA DDDDDDD BAAAA BBBBB DDDDDDD BAAAA AAAAA DDDDDDD BBAAA AAAABDDDDDDD BBAAA AAABA DDDDDDD BBAAA AAABB DDDDDDD BBAAA AABAA DDDDDDDBBAAA AABAB DDDDDDD BBAAA AABBA DDDDDDD BBAAA AABBB DDDDDDD BBAAA ABAAADDDDDDD BBAAA ABAAB DDDDDDD BBAAA ABABA DDDDDDD BBAAA ABABB DDDDDDDBBAAA ABBAA DDDDDDD BBAAA ABBAB DDDDDDD BBAAA ABBBA DDDDDDD BBAAA ABBBBDDDDDDD BBAAA BAAAA DDDDDDD BBAAA BAAAB DDDDDDD BBAAA BAABA DDDDDDDBBAAA BAABB DDDDDDD BBAAA BABAA DDDDDDD BBAAA BABAB DDDDDDD BBAAA BABBADDDDDDD BBAAA BABBB DDDDDDD BBAAA BBAAA DDDDDDD BBAAA BBAAB DDDDDDDBBAAA BBABA DDDDDDD BBAAA BBABB DDDDDDD BBAAA BBBAA DDDDDDD BBAAA BBBABDDDDDDD BBAAA BBBBA DDDDDDD BBAAA BBBBB DDDDDDD BBAAA AAAAA DDDDDDDBBBAA AAAAB DDDDDDD BBBAA AAABA DDDDDDD BBBAA AAABB DDDDDDD BBBAA AABAADDDDDDD BBBAA AABAB DDDDDDD BBBAA AABBA DDDDDDD BBBAA AABBB DDDDDDDBBBAA ABAAA DDDDDDD BBBAA ABAAB DDDDDDD BBBAA ABABA DDDDDDD BBBAA ABABBDDDDDDD BBBAA ABBAA DDDDDDD BBBAA ABBAB DDDDDDD BBBAA ABBBA DDDDDDDBBBAA ABBBB DDDDDDD BBBAA BAAAA DDDDDDD BBBAA BAAAB DDDDDDD BBBAA BAABADDDDDDD BBBAA BAABB DDDDDDD BBBAA BABAA DDDDDDD BBBAA BABAB DDDDDDDBBBAA BABBA DDDDDDD BBBAA BABBB DDDDDDD BBBAA BBAAA DDDDDDD BBBAA BBAABDDDDDDD BBBAA BBABA DDDDDDD BBBAA BBABB DDDDDDD BBBAA BBBAA DDDDDDDBBBAA BBBAB DDDDDDD BBBAA BBBBA DDDDDDD BBBAA BBBBB DDDDDDD BBBAA AAAAADDDDDDD BBBBA AAAAB DDDDDDD BBBBA AAABA DDDDDDD BBBBA AAABB DDDDDDDBBBBA AABAA DDDDDDD BBBBA AABAB DDDDDDD BBBBA AABBA DDDDDDD BBBBA AABBBDDDDDDD BBBBA ABAAA DDDDDDD BBBBA ABAAB DDDDDDD BBBBA ABABA DDDDDDDBBBBA ABABB DDDDDDD BBBBA ABBAA DDDDDDD BBBBA ABBAB DDDDDDD BBBBA ABBBADDDDDDD BBBBA ABBBB DDDDDDD BBBBA BAAAA DDDDDDD BBBBA BAAAB DDDDDDDBBBBA BAABA DDDDDDD BBBBA BAABB DDDDDDD BBBBA BABAA DDDDDDD BBBBA BABABDDDDDDD BBBBA BABBA DDDDDDD BBBBA BABBB DDDDDDD BBBBA BBAAA DDDDDDDBBBBA BBAAB DDDDDDD BBBBA BBABA DDDDDDD BBBBA BBABB DDDDDDD BBBBA BBBAADDDDDDD BBBBA BBBAB DDDDDDD BBBBA BBBBA DDDDDDD BBBBA BBBBB DDDDDDDBBBBA AAAAA DDDDDDD BBBBB AAAAB DDDDDDD BBBBB AAABA DDDDDDD BBBBB AAABBDDDDDDD BBBBB AABAA DDDDDDD BBBBB AABAB DDDDDDD BBBBB AABBA DDDDDDDBBBBB AABBB DDDDDDD BBBBB ABAAA DDDDDDD BBBBB ABAAB DDDDDDD BBBBB ABABADDDDDDD BBBBB ABABB DDDDDDD BBBBB ABBAA DDDDDDD BBBBB ABBAB DDDDDDDBBBBB ABBBA DDDDDDD BBBBB ABBBB DDDDDDD BBBBB BAAAA DDDDDDD BBBBB BAAABDDDDDDD BBBBB BAABA DDDDDDD BBBBB BAABB DDDDDDD BBBBB BABAA DDDDDDDBBBBB BABAB DDDDDDD BBBBB BABBA DDDDDDD BBBBB BABBB DDDDDDD BBBBB BBAAADDDDDDD BBBBB BBAAB DDDDDDD BBBBB BBABA DDDDDDD BBBBB BBABB DDDDDDDBBBBB BBBAA DDDDDDD BBBBB BBBAB DDDDDDD BBBBB BBBBA DDDDDDD BBBBB BBBBBDDDDDDD BBBBB

TABLE 12 Certain Gapmer Nucleoside Motifs 5′-wing Central gap 3′-wingregion region region AAAW DDDDDDDD BBA AABW DDDDDDDD BBA ABAW DDDDDDDDBBA ABBW DDDDDDDD BBA BAAW DDDDDDDD BBA BABW DDDDDDDD BBA BBAW DDDDDDDDBBA BBBW DDDDDDDD BBA ABB DDDDDDDD WAAA ABB DDDDDDDD WAAB ABB DDDDDDDDWABA ABB DDDDDDDD WABB ABB DDDDDDDD WBAA ABB DDDDDDDD WBAB ABB DDDDDDDDWBBA ABB DDDDDDDD WBBB AAAWW DDDDDDD BBA AABWW DDDDDDD BBA ABAWW DDDDDDDBBA ABBWW DDDDDDD BBA BAAWW DDDDDDD BBA BABWW DDDDDDD BBA BBAWW DDDDDDDBBA BBBWW DDDDDDD BBA ABB DDDDDDD WWAAA ABB DDDDDDD WWAAB ABB DDDDDDDWWABA ABB DDDDDDD WWABB ABB DDDDDDD WWBAA ABB DDDDDDD WWBAB ABB DDDDDDDWWBBA ABB DDDDDDD WWBBB AAAAW DDDDDDD BBA AAABW DDDDDDD BBA AABAWDDDDDDD BBA AABBW DDDDDDD BBA ABAAW DDDDDDD BBA ABABW DDDDDDD BBA ABBAWDDDDDDD BBA ABBBW DDDDDDD BBA BAAAW DDDDDDD BBA BAABW DDDDDDD BBA BABAWDDDDDDD BBA BABBW DDDDDDD BBA BBAAW DDDDDDD BBA BBABW DDDDDDD BBA BBBAWDDDDDDD BBA BBBBW DDDDDDD WAAAA ABB DDDDDDD WAAAB ABB DDDDDDD WAABA ABBDDDDDDD WAABB ABB DDDDDDD WABAA ABB DDDDDDD WABAB ABB DDDDDDD WABBA ABBDDDDDDD WABBB ABB DDDDDDD WBAAA ABB DDDDDDD WBAAB ABB DDDDDDD WBABA ABBDDDDDDD WBABB ABB DDDDDDD WBBAA ABB DDDDDDD WBBAB ABB DDDDDDD WBBBA ABBDDDDDDD WBBBB

wherein each A is a modified nucleoside of a first type, each B is amodified nucleoside of a second type and each W is a modified nucleosideor nucleobase of either the first type, the second type or a third type,each D is a nucleoside comprising an unmodified 2′ deoxy sugar moietyand unmodified nucleobase, and ^(N)D is modified nucleoside comprising amodified nucleobase and an unmodified 2′ deoxy sugar moiety.

In certain embodiments, each A comprises a modified sugar moiety. Incertain embodiments, each A comprises a 2′-substituted sugar moiety. Incertain embodiments, each A comprises a 2′-substituted sugar moietyselected from among F, ara-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each A comprises a bicyclic sugar moiety. In certainembodiments, each A comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each A comprises a modified nucleobase. In certainembodiments, each A comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certainembodiments, each A comprises an HNA. In certain embodiments, each Acomprises an F-HNA. In certain embodiments, each A comprises a5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me.

In certain embodiments, each B comprises a modified sugar moiety. Incertain embodiments, each B comprises a 2′-substituted sugar moiety. Incertain embodiments, each B comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each B comprises a bicyclic sugar moiety. In certainembodiments, each B comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each B comprises a modified nucleobase. In certainembodiments, each B comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. Incertain embodiments, each B comprises an HNA. In certain embodiments,each B comprises an F-HNA. In certain embodiments, each B comprises a5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me.

In certain embodiments, each C comprises a modified sugar moiety. Incertain embodiments, each C comprises a 2′-substituted sugar moiety. Incertain embodiments, each C comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each C comprises a 5′-substituted sugar moiety. In certainembodiments, each C comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me, and 5′-(R)-Me. In certain embodiments, each Ccomprises a bicyclic sugar moiety. In certain embodiments, each Ccomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises amodified nucleobase. In certain embodiments, each C comprises a modifiednucleobase selected from among 2-thio-thymidine and 5-propyne uridine.In certain embodiments, each C comprises a 2-thio-thymidine nucleoside.In certain embodiments, each C comprises an HNA. In certain embodiments,each C comprises an F-HNA.

In certain embodiments, each W comprises a modified sugar moiety. Incertain embodiments, each W comprises a 2′-substituted sugar moiety. Incertain embodiments, each W comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each W comprises a 5′-substituted sugar moiety. In certainembodiments, each W comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me, and 5′-(R)-Me. In certain embodiments, each Wcomprises a bicyclic sugar moiety. In certain embodiments, each Wcomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each W comprises asugar surrogate. In certain embodiments, each W comprises a sugarsurrogate selected from among HNA and F—HNA. In certain embodiments,each W comprises a 2-thio-thymidine nucleoside.

In certain embodiments, at least one of A or B comprises a bicyclicsugar moiety, and the other comprises a 2′-substituted sugar moiety. Incertain embodiments, one of A or B is an LNA nucleoside and the other ofA or B comprises a 2′-substituted sugar moiety. In certain embodiments,one of A or B is a cEt nucleoside and the other of A or B comprises a2′-substituted sugar moiety. In certain embodiments, one of A or B is anα-L-LNA nucleoside and the other of A or B comprises a 2′-substitutedsugar moiety. In certain embodiments, one of A or B is an LNA nucleosideand the other of A or B comprises a 2′-MOE sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-MOE sugar moiety. In certain embodiments, one of A or Bis an α-L-LNA nucleoside and the other of A or B comprises a 2′-MOEsugar moiety. In certain embodiments, one of A or B is an LNA nucleosideand the other of A or B comprises a 2′-F sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-F sugar moiety. In certain embodiments, one of A or B isan α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugarmoiety. In certain embodiments, one of A or B is an LNA nucleoside andthe other of A or B comprises a 2′-(ara)-F sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A orB is an α-L-LNA nucleoside and the other of A or B comprises a2′-(ara)-F sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-substituted sugar moiety. In certain embodiments, A is anLNA nucleoside and B comprises a 2′-substituted sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-substitutedsugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-substituted sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-MOE sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugarmoiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-MOE sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-F sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-F sugar moiety. In certain embodiments,A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certainembodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugarmoiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-(ara)-F sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugarmoiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-(ara)-F sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-MOE sugar moiety. In certainembodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugarmoiety. In certain embodiments, B is an α-L-LNA nucleoside and Acomprises a 2′-MOE sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-F sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-F sugar moiety. In certain embodiments,B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certainembodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugarmoiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-(ara)-F sugar moiety. In certainembodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugarmoiety. In certain embodiments, B is an α-L-LNA nucleoside and Acomprises a 2′-(ara)-F sugar moiety.

In certain embodiments, at least one of A or B comprises a bicyclicsugar moiety, another of A or B comprises a 2′-substituted sugar moietyand W comprises a modified nucleobase. In certain embodiments, one of Aor B is an LNA nucleoside, another of A or B comprises a 2′-substitutedsugar moiety, and W comprises a modified nucleobase. In certainembodiments, one of A or B is a cEt nucleoside, another of A or Bcomprises a 2′-substituted sugar moiety, and C comprises a modifiednucleobase. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-substituted sugar moiety,and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a modified nucleobase. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a modified nucleobase. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a modified nucleobase. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-substituted sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is an LNA nucleoside, another of A or B comprises a2′-substituted sugar moiety, and W comprises a 2-thio-thymidinenucleobase. In certain embodiments, one of A or B is a cEt nucleoside,another of A or B comprises a 2′-substituted sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a2′-substituted sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 2-thio-thymidine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 2-thio-thymidine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a 2-thio-thymidine nucleobase. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.In certain embodiments, one of A or B is an α-L-LNA nucleoside, anotherof A or B comprises a 2′-(ara)-F sugar moiety, and W comprises2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and C comprises a 5-propyne uridine nucleobase. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, andC comprises a 5-propyne uridine nucleobase. In certain embodiments, oneof A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 5-propyne uridinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is a cEt nucleoside, another of A or Bcomprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridinenucleobase. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises asugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is acEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is anα-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is an LNA nucleoside,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is a cEt nucleoside,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises asugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is acEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a sugar surrogate. In certain embodiments, one of A or Bis an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a HNA sugar surrogate. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a HNA sugar surrogate. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a sugar HNA surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises aHNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a HNA sugar surrogate. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises aF-HNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a F-HNA sugar surrogate. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a F-HNA sugar surrogate. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-Fsugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises aF-HNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a F-HNA sugar surrogate. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 5′-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-MeDNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-Fsugar moiety, and W comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a 5′-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of Aor B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of Aor B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.In certain embodiments, one of A or B is an α-L-LNA nucleoside, anotherof A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety.

In certain embodiments, at least two of A, B or W comprises a2′-substituted sugar moiety, and the other comprises a bicyclic sugarmoiety. In certain embodiments, at least two of A, B or W comprises abicyclic sugar moiety, and the other comprises a 2′-substituted sugarmoiety. In certain embodiments, a gapmer has a sugar motif other than:E-K-K-(D)₉-K-K-E; E-E-E-E-K-(D)₉-E-E-E-E-E; E-K-K-K-(D)₉-K-K-K-E;K-E-E-K-(D)₉-K-E-E-K; K-D-D-K-(D)₉-K-D-D-K; K-E-K-E-K-(D)₉-K-E-K-E-K;K-D-K-D-K-(D)₉-K-D-K-D-K; E-K-E-K-(D)₉-K-E-K-E;E-E-E-E-E-K-(D)₈-E-E-E-E-E; or E-K-E-K-E-(D)₉-E-K-E-K-E,E-E-E-K-K-(D)₇-E-E-K, E-K-E-K-K-K-(D)₇-K-E-K-E,E-K-E-K-E-K-(D)₇-K-E-K-E, wherein K is a nucleoside comprising a cEtsugar moiety and E is a nucleoside comprising a 2′-MOE sugar moiety.

In certain embodiments a gapmer comprises a A-(D)₄-A-(D)₄-A-(D)₄-AAmotif. In certain embodiments a gapmer comprises aB-(D)₄-A-(D)₄-A-(D)₄-AA motif. In certain embodiments a gapmer comprisesa A-(D)₄-B-(D)₄-A-(D)₄-AA motif. In certain embodiments a gapmercomprises a A-(D)₄-A-(D)₄-B-(D)₄-AA motif. In certain embodiments agapmer comprises a A-(D)₄-A-(D)₄-A-(D)₄-BA motif. In certain embodimentsa gapmer comprises a A-(D)₄-A-(D)₄-A-(D)₄-BB motif. In certainembodiments a gapmer comprises a K-(D)₄-K-(D)₄-K-(D)₄-K-E motif.

viii. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif, as described above for nucleoside motif. In suchembodiments, the internucleoside linkages in each of two wing regionsare different from the internucleoside linkages in the gap region. Incertain embodiments the internucleoside linkages in the wings arephosphodiester and the internucleoside linkages in the gap arephosphorothioate. The nucleoside motif is independently selected, sosuch oligonucleotides having a gapped internucleoside linkage motif mayor may not have a gapped nucleoside motif and if it does have a gappednucleoside motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present invention comprise a region of uniformlymodified internucleoside linkages. In certain such embodiments, theoligonucleotide comprises a region that is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide is uniformly linked by phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate and at least oneinternucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

In certain embodiments, oligonucleotides comprise one or moremethylphosphonate linkages. In certain embodiments, oligonucleotideshaving a gapmer nucleoside motif comprise a linkage motif comprising allphosphorothioate linkages except for one or two methylphosphonatelinkages. In certain embodiments, one methylphosphonate linkage is inthe central gap of an oligonucleotide having a gapmer nucleoside motif.

ix. Certain Modification Motifs

Modification motifs define oligonucleotides by nucleoside motif (sugarmotif and nucleobase motif) and linkage motif. For example, certainoligonucleotides have the following modification motif:

A_(s)A_(s)A_(s)D_(s)D_(s)D_(s)D_(s)(^(N)D)_(s)D_(s)D_(s)D_(s)D_(s)B_(s)B_(s)B;

wherein each A is a modified nucleoside comprising a 2′-substitutedsugar moiety; each D is an unmodified 2′-deoxynucleoside; each B is amodified nucleoside comprising a bicyclic sugar moiety; ^(N)D is amodified nucleoside comprising a modified nucleobase; and s is aphosphorothioate internucleoside linkage. Thus, the sugar motif is agapmer motif. The nucleobase modification motif is a single modifiednucleobase at 8^(th) nucleoside from the 5′-end. Combining the sugarmotif and the nucleobase modification motif, the nucleoside motif is aninterrupted gapmer where the gap of the sugar modified gapmer isinterrupted by a nucleoside comprising a modified nucleobase. Thelinkage motif is uniform phosphorothioate. The following non-limitingTable further illustrates certain modification motifs:

TABLE 13 Certain Modification Motifs 5′-wing Central gap 3′-wing regionregion region B_(s)B_(s)_(s)D_(s)D_(s)D_(s)D_(s)D_(s)D_(s)D_(s)D_(s)D_(s)A_(s)A_(s)A_(s)A_(s)A_(s)A_(s)A_(s)A AsBsBs DsDsDsDsDsDsDsDsDs BsBsAAsBsBs DsDsDsDs(^(N)D)sDsDsDsDs BsBsA AsBsBs DsDsDsDsAsDsDsDsDs BsBsAAsBsBs DsDsDsDsBsDsDsDsDs BsBsA AsBsBs DsDsDsDsWsDsDsDsDs BsBsA AsBsBsBsDsDsDsDsDsDsDsDsDs BsBsAsBsB AsBsBs DsDsDsDsDsDsDsDsDs BsBsAsBsBBsBsAsBsBs DsDsDsDsDsDsDsDsDs BsBsA AsBsBs DsDsDsDsDsDsDsDsDsBsBsAsBsBsBsB AsAsBsAsAs DsDsDsDsDsDsDsDsDs BsBsA AsAsAsBsAsAsDsDsDsDsDsDsDsDsDs BsBsA AsAsBsAsAs DsDsDsDsDsDsDsDsDs AsAsBsAsAAsAsAsBsAsAs DsDsDsDsDsDsDsDsDs AsAsBsAsAsA AsAsAsAsBsAsAsDsDsDsDsDsDsDsDsDs BsBsA AsBsAsBs DsDsDsDsDsDsDsDsDs BsAsBsA AsBsAsBsDsDsDsDsDsDsDsDsDs AsAsBsAsAs AsBsBs DsDsDsDsDsDsDsDsDs BsAsBsABsBsAsBsBsBsB DsDsDsDsDsDsDsDsDs BsAsBsA AsAsAsAsAs DsDsDsDsDsDsDsDsDsAsAsAsAsA AsAsAsAsAs DsDsDsDsDsDsDs AsAsAsAsA AsAsAsAsAsDsDsDsDsDsDsDsDsDs BsBsAsBsBsBsB AsAsAsBsBs DsDsDsDsDsDsDs BsBsAAsBsAsBs DsDsDsDsDsDsDsDs BsBsA AsBsAsBs DsDsDsDsDsDsDs AsAsAsBsBsAsAsAsAsBs DsDsDsDsDsDsDs BsAsAsAsA BsBs DsDsDsDsDsDsDsDs AsA AsAsDsDsDsDsDsDsDs AsAsAsAsAsAsAsA AsAsAs DsDsDsDsDsDsDs AsAsAsAsAsAsAAsAsAs DsDsDsDsDsDsDs AsAsAsAsAsA AsBs DsDsDsDsDsDsDs BsBsBsA AsBsBsBsDsDsDsDsDsDsDsDsDs BsA AsBs DsDsDsDsDsDsDsDsDs BsBsBsA AsAsAsBsBsDsDsDs(^(N)D)sDsDsDs BsBsAsAsA AsAsAsBsBs DsDsDsAsDsDsDs BsBsAsAsAAsAsAsBsBs DsDsDsBsDsDsDs BsBsAsAsA AsAsAsAsBs DsDsDsDsDsDsDs BsAsAsAsAAsAsBsBsBs DsDsDsDsDsDsDs BsBsBsAsA AsAsAsAsBs DsDsDsDsDsDsDs AsAsAsAsAsAsAsAsBsBs DsDsDsDsDsDsDs AsAsAsAsAs AsAsBsBsBs DsDsDsDsDsDsDsAsAsAsAsAs AsAsAsAsAs DsDsDsDsDsDsDs BsAsAsAsAs AsAsAsAsAsDsDsDsDsDsDsDs BsBsAsAsAs AsAsAsAsAs DsDsDsDsDsDsDs BsBsBsAsAs AsBsBsDsDsDsDs(^(N)D)s(^(N)D)sDsDsDs BsBsA AsBsBsDs(^(N)D)s(^(N)D)sDs(^(N)D)s(^(N)D)sDs(^(N)D)s(^(N)D)s BsBsA AsBsBsDs(^(N)D)sDsDsDsDsDsDsDs BsBsA AsBsBs DsDs(^(N)D)sDsDsDsDsDsDs BsBsAAsBsBs Ds(^(N)D)s(^(N)D)sDsDsDsDsDsDs BsBsA AsBsBs DsDs(D)zDsDsDsDsDsDsBsBsA AsBsBs Ds(D)zDsDsDsDsDsDsDs BsBsA AsBsBs (D)zDsDsDsDsDsDsDsDsBsBsA AsBsBs DsDsAsDsDsDsDsDsDs BsBsA AsBsBs DsDsBsDsDsDsDsDsDs BsBsAAsBsBs AsDsDsDsDsDsDsDsDs BsBsA AsBsBs BsDsDsDsDsDsDsDsDs BsBsA AsBsAsBsDsDs(D)zDsDsDsDsDsDs BsBsBsAsAs AsAsAsBsBs DsDs(^(N)D)sDsDsDsDsDsDs AsAAsBsBsBs Ds(D)zDsDsDsDsDsDsDs AsAsAsBsBs AsBsBs DsDsDsDsDsDsDsDs(D)zBsBsA AsAsBsBsBs DsDsDsAsDsDsDs BsBsBsAsA AsAsBsBsBs DsDsDsBsDsDsDsBsBsBsAsA AsBsAsBs DsDsDsAsDsDsDs BsBsAsBsBsBsB AsBsBsBsDsDsDsDs(D)zDsDsDsDs BsA AsAsBsBsBs DsDsAsAsDsDsDs BsBsA AsBsBsDsDsDsDs(D)zDsDsDsDs BsBsBsA BsBs DsDs(^(N)D)sDs(^(N)D)sDsDsDsDsBsBsAsBsBsBsB

wherein each A and B are nucleosides comprising differently modifiedsugar moieties, each D is a nucleoside comprising an unmodified 2′ deoxysugar moiety, each W is a modified nucleoside of either the first type,the second type or a third type, each ^(N)D is a modified nucleosidecomprising a modified nucleobase, s is a phosphorothioateinternucleoside linkage, and z is a non-phosphorothioate internucleosidelinkage.

In certain embodiments, each A comprises a modified sugar moiety. Incertain embodiments, each A comprises a 2′-substituted sugar moiety. Incertain embodiments, each A comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each A comprises a bicyclic sugar moiety. In certainembodiments, each A comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each A comprises a modified nucleobase. In certainembodiments, each A comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certainembodiments, each B comprises a modified sugar moiety. In certainembodiments, each B comprises a 2′-substituted sugar moiety. In certainembodiments, each B comprises a 2′-substituted sugar moiety selectedfrom among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certain embodiments,each B comprises a bicyclic sugar moiety. In certain embodiments, each Bcomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises amodified nucleobase. In certain embodiments, each B comprises a modifiednucleobase selected from among 2-thio-thymidine nucleoside and 5-propyneurindine nucleoside. In certain embodiments, each A comprises an HNA. Incertain embodiments, each A comprises an F-HNA.

In certain embodiments, each W comprises a modified sugar moiety. Incertain embodiments, each W comprises a 2′-substituted sugar moiety. Incertain embodiments, each W comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each W comprises a 5′-substituted sugar moiety. In certainembodiments, each W comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me, and 5′-(R)-Me. In certain embodiments, each Wcomprises a bicyclic sugar moiety. In certain embodiments, each Wcomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each W comprises asugar surrogate. In certain embodiments, each W comprises a sugarsurrogate selected from among HNA and F—HNA.

In certain embodiments, at least one of A or B comprises a bicyclicsugar moiety, and the other comprises a 2′-substituted sugar moiety. Incertain embodiments, one of A or B is an LNA nucleoside and the other ofA or B comprises a 2′-substituted sugar moiety. In certain embodiments,one of A or B is a cEt nucleoside and the other of A or B comprises a2′-substituted sugar moiety. In certain embodiments, one of A or B is anα-L-LNA nucleoside and the other of A or B comprises a 2′-substitutedsugar moiety. In certain embodiments, one of A or B is an LNA nucleosideand the other of A or B comprises a 2′-MOE sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-MOE sugar moiety. In certain embodiments, one of A or Bis an α-L-LNA nucleoside and the other of A or B comprises a 2′-MOEsugar moiety. In certain embodiments, one of A or B is an LNA nucleosideand the other of A or B comprises a 2′-F sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-F sugar moiety. In certain embodiments, one of A or B isan α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugarmoiety. In certain embodiments, one of A or B is an LNA nucleoside andthe other of A or B comprises a 2′-(ara)-F sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A orB is an α-L-LNA nucleoside and the other of A or B comprises a2′-(ara)-F sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-substituted sugar moiety. In certain embodiments, A is anLNA nucleoside and B comprises a 2′-substituted sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-substitutedsugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-substituted sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-MOE sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugarmoiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-MOE sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-F sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-F sugar moiety. In certain embodiments,A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certainembodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugarmoiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-(ara)-F sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugarmoiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-(ara)-F sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-MOE sugar moiety. In certainembodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugarmoiety. In certain embodiments, B is an α-L-LNA nucleoside and Acomprises a 2′-MOE sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-F sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-F sugar moiety. In certain embodiments,B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certainembodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugarmoiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-(ara)-F sugar moiety. In certainembodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugarmoiety. In certain embodiments, B is an α-L-LNA nucleoside and Acomprises a 2′-(ara)-F sugar moiety.

In certain embodiments, at least one of A or B comprises a bicyclicsugar moiety, another of A or B comprises a 2′-substituted sugar moietyand W comprises a modified nucleobase. In certain embodiments, one of Aor B is an LNA nucleoside, another of A or B comprises a 2′-substitutedsugar moiety, and W comprises a modified nucleobase. In certainembodiments, one of A or B is a cEt nucleoside, another of A or Bcomprises a 2′-substituted sugar moiety, and C comprises a modifiednucleobase. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-substituted sugar moiety,and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a modified nucleobase. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a modified nucleobase. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a modified nucleobase. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-substituted sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is an LNA nucleoside, another of A or B comprises a2′-substituted sugar moiety, and W comprises a 2-thio-thymidinenucleobase. In certain embodiments, one of A or B is a cEt nucleoside,another of A or B comprises a 2′-substituted sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a2′-substituted sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 2-thio-thymidine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 2-thio-thymidine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a 2-thio-thymidine nucleobase. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.In certain embodiments, one of A or B is an α-L-LNA nucleoside, anotherof A or B comprises a 2′-(ara)-F sugar moiety, and W comprises2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and C comprises a 5-propyne uridine nucleobase. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, andC comprises a 5-propyne uridine nucleobase. In certain embodiments, oneof A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 5-propyne uridinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is a cEt nucleoside, another of A or Bcomprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridinenucleobase. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises asugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is acEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is anα-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is an LNA nucleoside,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is a cEt nucleoside,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises asugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is acEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a sugar surrogate. In certain embodiments, one of A or Bis an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a HNA sugar surrogate. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a HNA sugar surrogate. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a sugar HNA surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises aHNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a HNA sugar surrogate. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises aF-HNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a F—HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a F-HNA sugar surrogate. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a F-HNA sugar surrogate. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-Fsugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises aF-HNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a F-HNA sugar surrogate. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 5′-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-MeDNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-Fsugar moiety, and W comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a 5′-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of Aor B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of Aor B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.In certain embodiments, one of A or B is an α-L-LNA nucleoside, anotherof A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety.

In certain embodiments, at least two of A, B or W comprises a2′-substituted sugar moiety, and the other comprises a bicyclic sugarmoiety. In certain embodiments, at least two of A, B or W comprises abicyclic sugar moiety, and the other comprises a 2′-substituted sugarmoiety.

d. Certain Overall Lengths

In certain embodiments, the present invention provides oligomericcompounds including oligonucleotides of any of a variety of ranges oflengths. In certain embodiments, the invention provides oligomericcompounds or oligonucleotides consisting of X to Y linked nucleosides,where X represents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X≦Y. For example, in certainembodiments, the invention provides oligomeric compounds which compriseoligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linkednucleosides. In embodiments where the number of nucleosides of anoligomeric compound or oligonucleotide is limited, whether to a range orto a specific number, the oligomeric compound or oligonucleotide may,nonetheless further comprise additional other substituents. For example,an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotideshaving 31 nucleosides, but, unless otherwise indicated, such anoligonucleotide may further comprise, for example one or moreconjugates, terminal groups, or other substituents. In certainembodiments, a gapmer oligonucleotide has any of the above lengths.

Further, where an oligonucleotide is described by an overall lengthrange and by regions having specified lengths, and where the sum ofspecified lengths of the regions is less than the upper limit of theoverall length range, the oligonucleotide may have additionalnucleosides, beyond those of the specified regions, provided that thetotal number of nucleosides does not exceed the upper limit of theoverall length range.

e. Certain Oligonucleotides

In certain embodiments, oligonucleotides of the present invention arecharacterized by their modification motif and overall length. In certainembodiments, such parameters are each independent of one another. Thus,unless otherwise indicated, each internucleoside linkage of anoligonucleotide having a gapmer sugar motif may be modified orunmodified and may or may not follow the gapmer modification pattern ofthe sugar modifications. For example, the internucleoside linkageswithin the wing regions of a sugar-gapmer may be the same or differentfrom one another and may be the same or different from theinternucleoside linkages of the gap region. Likewise, such sugar-gapmeroligonucleotides may comprise one or more modified nucleobaseindependent of the gapmer pattern of the sugar modifications. One ofskill in the art will appreciate that such motifs may be combined tocreate a variety of oligonucleotides. Herein if a description of anoligonucleotide or oligomeric compound is silent with respect to one ormore parameter, such parameter is not limited. Thus, an oligomericcompound described only as having a gapmer sugar motif without furtherdescription may have any length, internucleoside linkage motif, andnucleobase modification motif. Unless otherwise indicated, all chemicalmodifications are independent of nucleobase sequence.

f. Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by attachmentof one or more conjugate groups. In general, conjugate groups modify oneor more properties of the attached oligomeric compound including but notlimited to pharmacodynamics, pharmacokinetics, stability, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional conjugate linking moiety orconjugate linking group to a parent compound such as an oligomericcompound, such as an oligonucleotide. Conjugate groups includes withoutlimitation, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, phospholipids,biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine,fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groupshave been described previously, for example: cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al.,Ann N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

In certain embodiments, conjugate groups are directly attached tooligonucleotides in oligomeric compounds. In certain embodiments,conjugate groups are attached to oligonucleotides by a conjugate linkinggroup. In certain such embodiments, conjugate linking groups, including,but not limited to, bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Conjugate linkinggroups are useful for attachment of conjugate groups, such as chemicalstabilizing groups, functional groups, reporter groups and other groupsto selective sites in a parent compound such as for example anoligomeric compound. In general a bifunctional linking moiety comprisesa hydrocarbyl moiety having two functional groups. One of the functionalgroups is selected to bind to a parent molecule or compound of interestand the other is selected to bind essentially any selected group such aschemical functional group or a conjugate group. In some embodiments, theconjugate linker comprises a chain structure or an oligomer of repeatingunits such as ethylene glycol or amino acid units. Examples offunctional groups that are routinely used in a bifunctional linkingmoiety include, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In some embodiments, bifunctional linking moieties includeamino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double ortriple bonds), and the like.

Some nonlimiting examples of conjugate linking moieties includepyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Conjugate groups may be attached to either or both ends of anoligonucleotide (terminal conjugate groups) and/or at any internalposition.

In certain embodiments, conjugate groups are at the 3′-end of anoligonucleotide of an oligomeric compound. In certain embodiments,conjugate groups are near the 3′-end. In certain embodiments, conjugatesare attached at the 3′ end of an oligomeric compound, but before one ormore terminal group nucleosides. In certain embodiments, conjugategroups are placed within a terminal group. In certain embodiments, thepresent invention provides oligomeric compounds. In certain embodiments,oligomeric compounds comprise an oligonucleotide. In certainembodiments, an oligomeric compound comprises an oligonucleotide and oneor more conjugate and/or terminal groups. Such conjugate and/or terminalgroups may be added to oligonucleotides having any of the motifsdiscussed above. Thus, for example, an oligomeric compound comprising anoligonucleotide having region of alternating nucleosides may comprise aterminal group.

C. ANTISENSE COMPOUNDS

In certain embodiments, oligomeric compounds provided herein areantisense compounds. Such antisense compounds are capable of hybridizingto a target nucleic acid, resulting in at least one antisense activity.In certain embodiments, antisense compounds specifically hybridize toone or more target nucleic acid. In certain embodiments, a specificallyhybridizing antisense compound has a nucleobase sequence comprising aregion having sufficient complementarity to a target nucleic acid toallow hybridization and result in antisense activity and insufficientcomplementarity to any non-target so as to avoid non-specifichybridization to any non-target nucleic acid sequences under conditionsin which specific hybridization is desired (e.g., under physiologicalconditions for in vivo or therapeutic uses, and under conditions inwhich assays are performed in the case of in vitro assays).

In certain embodiments, the present invention provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary tothe target nucleic acid. In certain embodiments, such oligonucleotidesare 80% complementary to the target nucleic acid. In certainembodiments, an antisense compound comprises a region that is fullycomplementary to a target nucleic acid and is at least 80% complementaryto the target nucleic acid over the entire length of theoligonucleotide. In certain such embodiments, the region of fullcomplementarity is from 6 to 14 nucleobases in length.

a. Certain Antisense Activities and Mechanisms

In certain antisense activities, hybridization of an antisense compoundresults in recruitment of a protein that cleaves of the target nucleicacid. For example, certain antisense compounds result in RNase Hmediated cleavage of target nucleic acid. RNase H is a cellularendonuclease that cleaves the RNA strand of an RNA:DNA duplex. The “DNA”in such an RNA:DNA duplex, need not be unmodified DNA. In certainembodiments, the invention provides antisense compounds that aresufficiently “DNA-like” to elicit RNase H activity. Such DNA-likeantisense compounds include, but are not limited to gapmers havingunmodified deoxyfuronose sugar moieties in the nucleosides of the gapand modified sugar moieties in the nucleosides of the wings.

Antisense activities may be observed directly or indirectly. In certainembodiments, observation or detection of an antisense activity involvesobservation or detection of a change in an amount of a target nucleicacid or protein encoded by such target nucleic acid; a change in theratio of splice variants of a nucleic acid or protein; and/or aphenotypic change in a cell or animal.

In certain embodiments, compounds comprising oligonucleotides having agapmer nucleoside motif described herein have desirable propertiescompared to non-gapmer oligonucleotides or to gapmers having othermotifs. In certain circumstances, it is desirable to identify motifsresulting in a favorable combination of potent antisense activity andrelatively low toxicity. In certain embodiments, compounds of thepresent invention have a favorable therapeutic index (measure ofactivity divided by measure of toxicity).

b. Certain Selective Antisense Compounds

In certain embodiments, antisense compounds provided are selective for atarget relative to a non-target nucleic acid. In certain embodiments,the nucleobase sequences of the target and non-target nucleic acidsdiffer by no more than 4 differentiating nucleobases in the targetedregion. In certain embodiments, the nucleobase sequences of the targetand non-target nucleic acids differ by no more than 3 differentiatingnucleobases in the targeted region. In certain embodiments, thenucleobase sequences of the target and non-target nucleic acids differby no more than 2 differentiating nucleobases in the targeted region. Incertain embodiments, the nucleobase sequences of the target andnon-target nucleic acids differ by a single differentiating nucleobasein the targeted region. In certain embodiments, the target andnon-target nucleic acids are transcripts from different genes. Incertain embodiments, the target and non-target nucleic acids aredifferent alleles for the same gene. In certain embodiments, theintroduction of a mismatch between an antisense compound and anon-target nucleic acid may alter the RNase H cleavage site of a targetnucleic acid compared to a non-target nucleic acid. In certainembodiments, the target and non-target nucleic acids are notfunctionally related to one another (e.g., are transcripts fromdifferent genes). In certain embodiments, the target and not-targetnucleic acids are allelic variants of one another. In certainembodiments, the allelic variant contains a single nucleotidepolymorphism (SNP). In certain embodiments, a SNP is associated with amutant allele. In certain embodiments, a mutant SNP is associated with adisease. In certain embodiments a mutant SNP is associated with adisease, but is not causative of the disease. In certain embodiments,mRNA and protein expression of a mutant allele is associated withdisease.

Selectivity of antisense compounds is achieved, principally, bynucleobase complementarity. For example, if an antisense compound has nomismatches for a target nucleic acid and one or more mismatches for anon-target nucleic acid, some amount of selectivity for the targetnucleic acid will result. In certain embodiments, provided herein areantisense compounds with enhanced selectivity (i.e. the ratio ofactivity for the target to the activity for non-target is greater). Forexample, in certain embodiments, a selective nucleoside comprises aparticular feature or combination of features (e.g., chemicalmodification, motif, placement of selective nucleoside, and/orself-complementary region) that increases selectivity of an antisensecompound compared to an antisense compound not having that feature orcombination of features. In certain embodiments, such feature orcombination of features increases antisense activity for the target. Incertain embodiments, such feature or combination of features decreasesactivity for the target, but decreases activity for the non-target by agreater amount, thus resulting in an increase in selectivity.

Without being limited by mechanism, enhanced selectivity may result froma larger difference in the affinity of an antisense compound for itstarget compared to its affinity for the non-target and/or a largerdifference in RNase H activity for the resulting duplexes. For example,in certain embodiments, a selective antisense compound comprises amodified nucleoside at that same position as a differentiatingnucleobase (i.e., the selective nucleoside is modified). Thatmodification may increase the difference in binding affinity of theantisense compound for the target relative to the non-target. Inaddition or in the alternative, the chemical modification may increasethe difference in RNAse H activity for the duplex formed by theantisense compound and its target compared to the RNase activity for theduplex formed by the antisense compound and the non-target. For example,the modification may exaggerate a structure that is less compatible forRNase H to bind, cleave and/or release the non-target.

In certain embodiments, an antisense compound binds its intended targetto form a target duplex. In certain embodiments, RNase H cleaves thetarget nucleic acid of the target duplex. In certain such embodiments,there is a primary cleavage site between two particular nucleosides ofthe target nucleic acid (the primary target cleavage site), whichaccounts for the largest amount of cleavage of the target nucleic acid.In certain embodiments, there are one or more secondary target cleavagesites. In certain embodiments, the same antisense compound hybridizes toa non-target to form a non-target duplex. In certain such embodiments,the non-target differs from the target by a single nucleobase within thetarget region, and so the antisense compound hybridizes with a singlemismatch. Because of the mismatch, in certain embodiments, RNase Hcleavage of the non-target may be reduced compared to cleavage of thetarget, but still occurs. In certain embodiments, though, the primarysite of that cleavage of the non-target nucleic acid (primary non-targetcleavage site) is different from that of the target. That is; theprimary site is shifted due to the mismatch. In such a circumstance, onemay use a modification placed in the antisense compound to disrupt RNaseH cleavage at the primary non-target cleavage site. Such modificationwill result in reduced cleavage of the non-target, but will resultlittle or no decrease in cleavage of the target. In certain embodiments,the modification is a modified sugar, nucleobase and/or linkage.

In certain embodiments, the primary non-target cleavage site is towardsthe 5′-end of the antisense compound, and the 5′-end of an antisensecompound may be modified to prevent RNaseH cleavage. In this manner, itis thought that one having skill in the art may modify the 5′-end of anantisense compound, or modify the nucleosides in the gap region of the5′-end of the antisense compound, or modify the 3′-most 5′-regionnucleosides of the antisense compound to selectively inhibit RNaseHcleavage of the non-target nucleic acid duplex while retaining RNase Hcleavage of the target nucleic acid duplex. In certain embodiments, 1-3of the 3′-most 5′-region nucleosides of the antisense compound comprisesa bicyclic sugar moiety.

For example, in certain embodiments the target nucleic acid may have anallelic variant, e.g. a non-target nucleic acid, containing a singlenucleotide polymorphism. An antisense compound may be designed having asingle nucleobase mismatch from the non-target nucleic acid, but whichhas full complementarity to the target nucleic acid. The mismatchbetween the antisense compound and the non-target nucleic acid maydestabilize the antisense compound non-target nucleic acid duplex, andconsequently the cleavage site of RNaseH may shift upstream towards the5′-end of the antisense compound. Modification of the 5′-end of theantisense compound or the gap region near the 5′-end of the antisensecompound, or one or more of the 3′-most nucleosides of the 5′-wingregion, will then prevent RNaseH cleavage of the non-target nucleicacid. Since the target nucleic acid is fully complementary to theantisense compound, the antisense compound and the target nucleic acidwill form a more stabilized antisense compound-target nucleic acidduplex and the cleavage site of RnaseH will be more downstream, towardsthe 3′ end of the antisense compound. Accordingly, modifications at the5′-end of the antisense compound will prevent RNaseH cleavage of thenon-target nucleic acid, but will not substantially effect RNaseHcleavage of the target nucleic acid, and selectivity between a targetnucleic acid and its allelic variant may be achieved. In certainembodiments, one or more of the 3′-most nucleosides of the 5′-wingregion comprises a bicyclic sugar moiety. In certain embodiments, one ormore of the 3′-most nucleosides of the 5′-wing region comprises abicyclic sugar moiety selected from cEt and LNA. In certain embodiments,one or more of the 3′-most nucleosides of the 5′-wing region comprisescEt. In certain embodiments, one or more of the 3′-most nucleosides ofthe 5′-wing region comprises LNA.

In certain embodiments, the introduction of a mismatch between anantisense compound and a target nucleic acid may alter the RNase Hcleavage site of a target nucleic acid compared to a non-target nucleicacid by shifting the RNaseH cleavage site downstream from the mismatchsite and towards the 3′-end of the antisense compound. In certainembodiments where the cleavage site of a target nucleic acid compared toa non-target nucleic acid has shifted downstream towards the 3′-end ofthe antisense compound, the 3′-end of an antisense compound may bemodified to prevent RNaseH cleavage. In this manner, it is thought thatone having skill in the art may modify the 3′-end of an antisensecompound, or modify the nucleosides in the gap region near the 3′-end ofantisense compound, to selectively inhibit RNaseH cleavage of thenon-target nucleic acid while retaining RNase H cleavage of the targetnucleic acid.

For example, in certain embodiments the target nucleic acid may have anallelic variant, e.g. a non-target nucleic acid, containing a singlenucleotide polymorphism. An antisense compound may be designed having asingle nucleobase mismatch from the non-target nucleic acid, but whichhas full complementarity to target nucleic acid. The mismatch betweenthe antisense compound and the non-target nucleic acid may destabilizethe antisense compound-non-target nucleic acid duplex, and consequentlythe cleavage site of RNaseH may shift downstream towards the 3′-end ofthe antisense compound. Modification of the 3′-end of the antisensecompound, or one or more of the 5′-most nucleosides of the 3′-wingregion, or the gap region of the antisense compound near the 3′-end willthen prevent RNaseH cleavage of the non-target nucleic acid. Since thetarget nucleic acid is fully complementary to the antisense compound,the antisense compound and the target nucleic acid will form a morestabilized antisense compound-target nucleic acid duplex and thecleavage site of RnaseH will be more upstream, towards the 5′ end of theantisense compound. Accordingly, modifications at the 3′-end of theantisense compound will prevent RNaseH cleavage of the non-targetnucleic acid, but will not substantially effect RNaseH cleavage of thetarget nucleic acid, and selectivity between a target nucleic acid andits allelic variant may be achieved. In certain embodiments, one or moreof the 5′-most nucleosides of the 3′-wing region comprises a bicyclicsugar moiety. In certain embodiments, one or more of the 5′-mostnucleosides of the 3′-wing region comprises a bicyclic sugar moietyselected from cEt and LNA. In certain embodiments, one or more of the5′-most nucleosides of the 3′-wing region comprises cEt. In certainembodiments, one or more of the 5′-most nucleosides of the 3′-wingregion comprises LNA.

In certain embodiments, the selectivity of antisense compounds havingcertain gaps, e.g. gaps of 7 nucleosides or longer, may be improved bythe addition of one or more bicyclic nucleosides at the 3′-most 5′-wingnucleoside. In certain embodiments, the selectivity of antisensecompounds having certain gaps, e.g. gaps of 7 nucleosides or longer, maybe improved by the addition of two or more bicyclic nucleosides at the3′-most 5′-wing nucleoside. In certain embodiments, the selectivity ofantisense compounds having certain gaps, e.g. gaps of 7 nucleosides orlonger, may be improved by the addition of one bicyclic nucleoside atthe 3′-most 5′-wing nucleoside. In certain embodiments, the selectivityof antisense compounds having certain gaps, e.g. gaps of 7 nucleosidesor longer, may be improved by the addition of two bicyclic nucleosidesat the 3′-most 5′-wing nucleoside. In certain embodiments, theselectivity of antisense compounds having certain gaps, e.g. gaps of 7nucleosides or longer, may be improved by the addition of three bicyclicnucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments,the selectivity of antisense compounds having certain gaps, e.g. gaps of7 nucleosides or longer, may be improved by the addition of fourbicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certainembodiments, the selectivity of antisense compounds having certain gaps,e.g. gaps of 7 nucleosides or longer, may be improved by the addition offive bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certainembodiments discussed above, the bicyclic nucleosides at the 3′-most5′-wing nucleoside are selected from among cEt, cMOE, LNA, α-LNA, ENAand 2′-thio LNA. In certain embodiments discussed above, the bicyclicnucleosides at the 3′-most 5′-wing nucleoside comprise cEt. In certainembodiments discussed above, the bicyclic nucleosides at the 3′-most5′-wing nucleoside comprise LNA.

In certain embodiments, the selectivity of antisense compounds havingcertain gaps, e.g. gaps of 7 nucleosides or longer, may be improved bythe addition of one or more bicyclic nucleosides at the 3′-most 5′-wingnucleoside and the addition of one or more bicyclic nucleosides at the5′-most 3′-wing nucleoside. In certain embodiments, the selectivity ofantisense compounds having certain gaps, e.g. gaps of 7 nucleosides orlonger, may be improved by the addition of two or more bicyclicnucleosides at the 3′-most 5′-wing nucleoside and the addition of one ormore bicyclic nucleosides at the 5′-most 3′-wing nucleoside. In certainembodiments, the selectivity of antisense compounds having certain gaps,e.g. gaps of 7 nucleosides or longer, may be improved by the addition ofone bicyclic nucleoside at the 3′-most 5′-wing nucleoside and theaddition of one or more bicyclic nucleosides at the 5′-most 3′-wingnucleoside. In certain embodiments, the selectivity of antisensecompounds having certain gaps, e.g. gaps of 7 nucleosides or longer, maybe improved by the addition of two bicyclic nucleosides at the 3′-most5′-wing nucleoside and the addition of one or more bicyclic nucleosidesat the 5′-most 3′-wing nucleoside. In certain embodiments, theselectivity of antisense compounds having certain gaps, e.g. gaps of 7nucleosides or longer, may be improved by the addition of three bicyclicnucleosides at the 3′-most 5′-wing nucleoside and the addition of one ormore bicyclic nucleosides at the 5′-most 3′-wing nucleoside. In certainembodiments, the selectivity of antisense compounds having certain gaps,e.g. gaps of 7 nucleosides or longer, may be improved by the addition offour bicyclic nucleosides at the 3′-most 5′-wing nucleoside and theaddition of one or more bicyclic nucleosides at the 5′-most 3′-wingnucleoside. In certain embodiments, the selectivity of antisensecompounds having certain gaps, e.g. gaps of 7 nucleosides or longer, maybe improved by the addition of four bicyclic nucleosides at the 3′-most5′-wing nucleoside and the addition of one or more bicyclic nucleosidesat the 5′-most 3′-wing nucleoside.

In certain embodiments, the selectivity of antisense compounds havingcertain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved bythe addition of one or more bicyclic nucleosides at the 3′-most 5′-wingnucleoside. In certain embodiments, the selectivity of antisensecompounds having certain gaps, e.g. gaps of 7 nucleosides or shorter,may be improved by the addition of two or more bicyclic nucleosides atthe 3′-most 5′-wing nucleoside. In certain embodiments, the selectivityof antisense compounds having certain gaps, e.g. gaps of 7 nucleosidesor shorter, may be improved by the addition of one bicyclic nucleosideat the 3′-most 5′-wing nucleoside. In certain embodiments, theselectivity of antisense compounds having certain gaps, e.g. gaps of 7nucleosides or shorter, may be improved by the addition of two bicyclicnucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments,the selectivity of antisense compounds having certain gaps, e.g. gaps of7 nucleosides or shorter, may be improved by the addition of threebicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certainembodiments, the selectivity of antisense compounds having certain gaps,e.g. gaps of 7 nucleosides or shorter, may be improved by the additionof four bicyclic nucleosides at the 3′-most 5′-wing nucleoside. Incertain embodiments, the selectivity of antisense compounds havingcertain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved bythe addition of five bicyclic nucleosides at the 3′-most 5′-wingnucleoside. In certain embodiments discussed above, the bicyclicnucleosides at the 3′-most 5′-wing nucleoside are selected from amongcEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA. In certain embodimentsdiscussed above, the bicyclic nucleosides at the 3′-most 5′-wingnucleoside comprise cEt. In certain embodiments discussed above, thebicyclic nucleosides at the 3′-most 5′-wing nucleoside comprise LNA.

Antisense compounds having certain specified motifs have enhancedselectivity, including, but not limited to motifs described above. Incertain embodiments, enhanced selectivity is achieved byoligonucleotides comprising any one or more of:

a modification motif comprising a long 5′-wing (longer than 5, 6, or 7nucleosides);

a modification motif comprising a long 3′-wing (longer than 5, 6, or 7nucleosides);

a modification motif comprising a short gap region (shorter than 8, 7,or 6 nucleosides); and

a modification motif comprising an interrupted gap region (having nouninterrupted stretch of unmodified 2′-deoxynucleosides longer than 7, 6or 5).

i. Certain Selective Nucleobase Sequence Elements

In certain embodiments, selective antisense compounds comprisenucleobase sequence elements. Such nucleobase sequence elements areindependent of modification motifs. Accordingly, oligonucleotides havingany of the motifs (modification motifs, nucleoside motifs, sugar motifs,nucleobase modification motifs, and/or linkage motifs) may also compriseone or more of the following nucleobase sequence elements.

ii. Alignment of Differentiating Nucleobase/Target-Selective Nucleoside

In certain embodiments, a target region and a region of a non-targetnucleic acid differ by 1-4 differentiating nucleobase. In suchembodiments, selective antisense compounds have a nucleobase sequencethat aligns with the non-target nucleic acid with 1-4 mismatches. Anucleoside of the antisense compound that corresponds to adifferentiating nucleobase of the target nucleic acid is referred toherein as a target-selective nucleoside. In certain embodiments,selective antisense compounds having a gapmer motif align with anon-target nucleic acid, such that a target-selective nucleoside ispositioned in the gap. In certain embodiments, a target-selectivenucleoside is the 1^(st) nucleoside of the gap from the 5′ end. Incertain embodiments, a target-selective nucleoside is the 2^(nd)nucleoside of the gap from the 5′ end. In certain embodiments, atarget-selective nucleoside is the 3^(rd) nucleoside of the gap from the5′-end. In certain embodiments, a target-selective nucleoside is the4^(th) nucleoside of the gap from the 5′-end. In certain embodiments, atarget-selective nucleoside is the 5^(th) nucleoside of the gap from the5′-end. In certain embodiments, a target-selective nucleoside is the6^(rd) nucleoside of the gap from the 5′-end. In certain embodiments, atarget-selective nucleoside is the 8^(th) nucleoside of the gap from the3′-end. In certain embodiments, a target-selective nucleoside is the7^(th) nucleoside of the gap from the 3′-end. In certain embodiments, atarget-selective nucleoside is the 6^(th) nucleoside of the gap from the3′-end. In certain embodiments, a target-selective nucleoside is the5^(th) nucleoside of the gap from the 3′-end. In certain embodiments, atarget-selective nucleoside is the 4^(th) nucleoside of the gap from the3′-end. In certain embodiments, a target-selective nucleoside is the3^(rd) nucleoside of the gap from the 3′-end. In certain embodiments, atarget-selective nucleoside is the 2^(nd) nucleoside of the gap from the3′-end.

In certain embodiments, a target-selective nucleoside comprises amodified nucleoside. In certain embodiments, a target-selectivenucleoside comprises a modified sugar. In certain embodiments, atarget-selective nucleoside comprises a sugar surrogate. In certainembodiments, a target-selective nucleoside comprises a sugar surrogateselected from among HNA and F-HNA. In certain embodiments, atarget-selective nucleoside comprises a 2′-substituted sugar moiety. Incertain embodiments, a target-selective nucleoside comprises a2′-substituted sugar moiety selected from among MOE, F and (ara)-F. Incertain embodiments, a target-selective nucleoside comprises a5′-substituted sugar moiety. In certain embodiments, a target-selectivenucleoside comprises a 5′-substituted sugar moiety selected from5′-(R)-Me DNA. In certain embodiments, a target-selective nucleosidecomprises a bicyclic sugar moiety. In certain embodiments, atarget-selective nucleoside comprises a bicyclic sugar moiety selectedfrom among cEt, and α-L-LNA. In certain embodiments, a target-selectivenucleoside comprises a modified nucleobase. In certain embodiments, atarget-selective nucleoside comprises a modified nucleobase selectedfrom among 2-thio-thymidine and 5-propyne uridine.

iii. Mismatches to the Target Nucleic Acid

In certain embodiments, selective antisense compounds comprise one ormore mismatched nucleobases relative to the target nucleic acid. Incertain such embodiments, antisense activity against the target isreduced by such mismatch, but activity against the non-target is reducedby a greater amount. Thus, in certain embodiments selectivity isimproved. Any nucleobase other than the differentiating nucleobase issuitable for a mismatch. In certain embodiments, however, the mismatchis specifically positioned within the gap of an oligonucleotide having agapmer motif. In certain embodiments, a mismatch relative to the targetnucleic acid is at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the 5′-endof the gap region. In certain embodiments, a mismatch relative to thetarget nucleic acid is at positions 9, 8, 7, 6, 5, 4, 3, 2, 1 of theantisense compounds from the 3′-end of the gap region. In certainembodiments, a mismatch relative to the target nucleic acid is atpositions 1, 2, 3, or 4 of the antisense compounds from the 5′-end ofthe wing region. In certain embodiments, a mismatch relative to thetarget nucleic acid is at positions 4, 3, 2, or 1 of the antisensecompounds from the 3′-end of the wing region.

iv. Self Complementary Regions

In certain embodiments, selective antisense compounds comprise a regionthat is not complementary to the target. In certain embodiments, suchregion is complementary to another region of the antisense compound.Such regions are referred to herein as self-complementary regions. Forexample, in certain embodiments, an antisense compound has a firstregion at one end that is complementary to a second region at the otherend. In certain embodiments, one of the first and second regions iscomplementary to the target nucleic acid. Unless the target nucleic acidalso includes a self-complementary region, the other of the first andsecond region of the antisense compound will not be complementary to thetarget nucleic acid. For illustrative purposes, certain antisensecompounds have the following nucleobase motif:

ABCXXXXXXXXXC′B′A′; ABCXXXXXXX(X/C′)(X/B′)(X/A′);(X/A)(X/B)(X/C)XXXXXXXXXC′B′A′where each of A, B, and C are any nucleobase; A′, B′, and C′ are thecomplementary bases to A, B, and C, respectively; each X is a nucleobasecomplementary to the target nucleic acid; and two letters in parentheses(e.g., (X/C′)) indicates that the nucleobase is complementary to thetarget nucleic acid and to the designated nucleoside within theantisense oligonucleotide.

Without being bound to any mechanism, in certain embodiments, suchantisense compounds are expected to form self-structure, which isdisrupted upon contact with a target nucleic acid. Contact with anon-target nucleic acid is expected to disrupt the self-structure to alesser degree, thus increasing selectivity compared to the sameantisense compound lacking the self-complementary regions.

v. Combinations of Features

Though it is clear to one of skill in the art, the above motifs andother elements for increasing selectivity may be used alone or incombination. For example, a single antisense compound may include anyone, two, three, or more of: self-complementary regions, a mismatchrelative to the target nucleic acid, a short nucleoside gap, aninterrupted gap, and specific placement of the selective nucleoside.

D. CERTAIN SHORT GAP ANTISENSE COMPOUNDS

In certain embodiments, an antisense compound of interest may modulatethe expression of a target nucleic acid but possess undesirableproperties. In certain embodiments, for example, an antisense compoundof interest may have an undesirably high affinity for one or morenon-target nucleic acids. In certain embodiments, whether as a result ofsuch affinity for one or more non-target nucleic acid or by some othermechanism, an antisense compound of interest may produce undesirableincreases in ALT and/or AST levels when administered to an animal. Incertain embodiments, such an antisense compound of interest may produceundesirable increases in organ weight.

In certain such embodiments wherein an antisense compound of interesteffectively modulates the expression of a target nucleic acid, butpossess one or more undesirable properties, a person having skill in theart may selectively incorporate one or more modifications into theantisense compound of interest that retain some or all of the desiredproperty of effective modulation of expression of a target nucleic acidwhile reducing one or more of the antisense compound's undesirableproperties. In certain embodiments, the present invention providesmethods of altering such an antisense compound of interest to form animproved antisense compound. In certain embodiments, altering the numberof nucleosides in the 5′-region, the 3′-region, and/or the centralregion of such an antisense compound of interest results in improvedproperties. For example, in certain embodiments, one may alter themodification state of one or more nucleosides at or near the 5′-end ofthe central region. Having been altered, those nucleosides may then becharacterized as being part of the 5′-region. Thus, in such embodiments,the overall number of nucleosides of the 5′-region is increased and thenumber of nucleosides in the central region is decreased. For example,an antisense compound having a modification motif of 3-10-3 could bealtered to result in an improved antisense compound having amodification motif of 4-9-3 or 5-8-3. In certain embodiments, themodification state of one or more of nucleosides at or near the 3′-endof the central region may likewise be altered. In certain embodiments,the modification of one or more of the nucleosides at or near the 5′-endand the 3′-end of the central region may be altered. In such embodimentsin which one or more nucleosides at or near the 5′-end and the 3′-end ofthe central region is altered the central region becomes shorterrelative to the central region of the original antisense compound ofinterest. In such embodiments, the modifications to the one or morenucleosides that had been part of the central region are the same as oneor more modification that had been present in the 5′-region and/or the3′-region of the original antisense compound of interest. In certainembodiments, the improved antisense compound having a shortened centralregion may retain its ability to effectively modulate the expression ofa target nucleic acid, but not possess some or all of the undesirableproperties possessed by antisense compound of interest having a longercentral region. In certain embodiments, reducing the length of thecentral region reduces affinity for off-target nucleic acids. In certainembodiments, reducing the length of the central region results inreduced cleavage of non-target nucleic acids by RNase H. In certainembodiments, reducing the length of the central region does not produceundesirable increases in ALT levels. In certain embodiments, reducingthe length of the central region does not produce undesirable increasesin AST levels. In certain embodiments, reducing the length of thecentral region does not produce undesirable increases organ weights.

In certain embodiments it is possible to retain the same nucleobasesequence and overall length of an antisense compound while decreasingthe length of the central region. In certain embodiments retaining thesame nucleobase sequence and overall length of an antisense compoundwhile decreasing the length of the central region ameliorates one ormore undesirable properties of an antisense compound. In certainembodiments retaining the same nucleobase sequence and overall length ofan antisense compound while decreasing the length of the central regionameliorates one or more undesirable properties of an antisense compoundbut does not substantially affect the ability of the antisense compoundto modulate expression of a target nucleic acid. In certain suchembodiments, two or more antisense compounds would have the same overalllength and nucleobase sequence, but would have a different centralregion length, and different properties. In certain embodiments, thelength of the central region is 9 nucleobases. In certain embodiments,the length of the central region is 8 nucleobases. In certainembodiments, the length of the central region is 7 nucleobases. Incertain embodiments, the central region consists of unmodifieddeoxynucleosides. In certain embodiments, the length of the centralregion can be decreased by increasing the length of the 5′-region, the3′-region, or both the 5′-region and the 3′-region.

In certain embodiments, the length of the central region can bedecreased by increasing the length of the 5′-region with modifiednucleosides. In certain embodiments, the length of the central regioncan be decreased by increasing the length of the 5′-region with modifiednucleosides. In certain embodiments, the length of the central regioncan be decreased by increasing the length of the 5′-region with modifiednucleosides comprising a bicyclic sugar moiety selected from among: cEt,cMOE, LNA, α-LNA, ENA and 2′-thio LNA. In certain embodiments, thelength of the central region can be decreased by increasing the lengthof the 5′-region with a cEt substituted sugar moiety.

In certain embodiments, the length of the central region can bedecreased by increasing the length of the 5′-region with modifiednucleosides. In certain embodiments, the length of the central regioncan be decreased by increasing the length of the 5′-region with modifiednucleosides. In certain embodiments, the length of the central regioncan be decreased by increasing the length of the 5′-region with modifiednucleosides comprising a bicyclic sugar moiety comprising a 2′substituent selected from among: a halogen, OCH₃, OCF₃, OCH₂CH₃,OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃ (MOE), O(CH₂)₂—O(CH₂)₂—N(CH₃)₂,OCH₂C(═O)—N(H)CH₃, OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, andOCH₂—N(H)—C(═NH)NH₂. In certain embodiments, the length of the centralregion can be decreased by increasing the length of the 5′-region with2′-O(CH₂)₂—OCH₃ (MOE) substituted sugar moiety.

In certain embodiments, the length of the central region can bedecreased by increasing the length of the 3′-region with modifiednucleosides. In certain embodiments, the length of the central regioncan be decreased by increasing the length of the 3′-region with modifiednucleosides. In certain embodiments, the length of the central regioncan be decreased by increasing the length of the 3′-region with modifiednucleosides comprising a bicyclic sugar moiety selected from among: cEt,cMOE, LNA, α-LNA, ENA and 2′-thio LNA. In certain embodiments, thelength of the central region can be decreased by increasing the lengthof the 3′-region with a cEt substituted sugar moiety.

In certain embodiments, the length of the central region can bedecreased by increasing the length of the 3′-region with modifiednucleosides. In certain embodiments, the length of the central regioncan be decreased by increasing the length of the 3′-region with modifiednucleosides. In certain embodiments, the length of the central regioncan be decreased by increasing the length of the 3′-region with modifiednucleosides comprising a bicyclic sugar moiety comprising a 2′substituent selected from among: a halogen, OCH₃, OCF₃, OCH₂CH₃,OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃ (MOE), O(CH₂)₂—O(CH₂)₂—N(CH₃)₂,OCH₂C(═O)—N(H)CH₃, OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, andOCH₂—N(H)—C(═NH)NH₂. In certain embodiments, the length of the centralregion can be decreased by increasing the length of the 3′-region with2′-O(CH₂)₂—OCH₃ (MOE) substituted sugar moiety.

In certain embodiments, the length of the central region can bedecreased by increasing the length of the 5′-region with modifiednucleosides and increasing the length of the 3′-region with modifiednucleosides.

E. CERTAIN TARGET NUCLEIC ACIDS

In certain embodiments, antisense compounds comprise or consist of anoligonucleotide comprising a region that is complementary to a targetnucleic acid. In certain embodiments, the target nucleic acid is anendogenous RNA molecule. In certain embodiments, the target nucleic acidis a non-coding RNA. In certain such embodiments, the target non-codingRNA is selected from: a long-non-coding RNA, a short non-coding RNA, anintronic RNA molecule, a snoRNA, a scaRNA, a microRNA (includingpre-microRNA and mature microRNA), a ribosomal RNA, and promoterdirected RNA. In certain embodiments, the target nucleic acid encodes aprotein. In certain such embodiments, the target nucleic acid isselected from: an mRNA and a pre-mRNA, including intronic, exonic anduntranslated regions. In certain embodiments, oligomeric compounds areat least partially complementary to more than one target nucleic acid.For example, antisense compounds of the present invention may mimicmicroRNAs, which typically bind to multiple targets.

In certain embodiments, the target nucleic acid is a nucleic acid otherthan a mature mRNA. In certain embodiments, the target nucleic acid is anucleic acid other than a mature mRNA or a microRNA. In certainembodiments, the target nucleic acid is a non-coding RNA other than amicroRNA. In certain embodiments, the target nucleic acid is anon-coding RNA other than a microRNA or an intronic region of apre-mRNA. In certain embodiments, the target nucleic acid is a longnon-coding RNA. In certain embodiments, the target RNA is an mRNA. Incertain embodiments, the target nucleic acid is a pre-mRNA. In certainsuch embodiments, the target region is entirely within an intron. Incertain embodiments, the target region spans an intron/exon junction. Incertain embodiments, the target region is at least 50% within an intron.In certain embodiments, the target nucleic acid is selected from amongnon-coding RNA, including exonic regions of pre-mRNA. In certainembodiments, the target nucleic acid is a ribosomal RNA (rRNA). Incertain embodiments, the target nucleic acid is a non-coding RNAassociated with splicing of other pre-mRNAs. In certain embodiments, thetarget nucleic acid is a nuclear-retained non-coding RNA.

In certain embodiments, antisense compounds described herein arecomplementary to a target nucleic acid comprising a single-nucleotidepolymorphism. In certain such embodiments, the antisense compound iscapable of modulating expression of one allele of the single-nucleotidepolymorphism-containing-target nucleic acid to a greater or lesserextent than it modulates another allele. In certain embodiments anantisense compound hybridizes to a single-nucleotidepolymorphism-containing-target nucleic acid at the single-nucleotidepolymorphism site. In certain embodiments, the target nucleic acid is aHuntingtin gene transcript. In certain embodiments, the target nucleicacid is a single-nucleotide polymorphism-containing-target nucleic acidof a Huntingtin gene transcript. In certain embodiments, the targetnucleic acid is not a Huntingtin gene transcript. In certainembodiments, the target nucleic acid is a single-nucleotidepolymorphism-containing-target nucleic acid of a gene transcript otherthan Huntingtin. In certain embodiments, the target nucleic acid is anynucleic acid other than a Huntingtin gene transcript.

a. Single-Nucleotide Polymorphism

In certain embodiments, the invention provides selective antisensecompounds that have greater activity for a target nucleic acid than fora homologous or partially homologous non-target nucleic acid. In certainsuch embodiments, the target and non-target nucleic acids are notfunctionally related to one another (e.g., are transcripts fromdifferent genes). In certain embodiments, the target and not-targetnucleic acids are allelic variants of one another. Certain embodimentsof the present invention provide methods, compounds, and compositionsfor selectively inhibiting mRNA and protein expression of an allelicvariant of a particular gene or DNA sequence. In certain embodiments,the allelic variant contains a single nucleotide polymorphism (SNP). Incertain embodiments, a SNP is associated with a mutant allele. Incertain embodiments, a mutant SNP is associated with a disease. Incertain embodiments a mutant SNP is associated with a disease, but isnot causative of the disease. In certain embodiments, mRNA and proteinexpression of a mutant allele is associated with disease.

In certain embodiments, the expressed gene product of a mutant alleleresults in aggregation of the mutant proteins causing disease. Incertain embodiments, the expressed gene product of a mutant alleleresults in gain of function causing disease. In certain embodiments,genes with an autosomal dominant mutation resulting in a toxic gain offunction of the protein are the APP gene encoding amyloid precursorprotein involved in Alzheimer's disease (Gene, 371: 68, 2006); the PrPgene encoding prion protein involved in Creutzfeldt-Jakob disease and infatal familial insomnia (Nat. Med. 1997, 3: 1009); GFAP gene encodingglial fibrillary acidic protein involved in Alexander disease (J.Neurosci. 2006, 26:111623); alpha-synuclein gene encodingalpha-synuclein protein involved in Parkinson's disease (J. Clin.Invest. 2003, 111: 145); SOD-1 gene encoding the SOD-1 protein involvedin amyotrophic lateral sclerosis (Science 1998, 281: 1851); atrophin-1gene encoding atrophin-1 protein involved in dentato-rubral andpallido-luysian atrophy (DRPA) (Trends Mol. Med. 2001, 7: 479); SCA1gene encoding ataxin-1 protein involved in spino-cerebellar ataxia-1(SCA1) (Protein Sci. 2003, 12: 953); PLP gene encoding proteolipidprotein involved in Pelizaeus-Merzbacher disease (NeuroMol. Med. 2007,4: 73); DYT1 gene encoding torsinA protein involved in Torsion dystonia(Brain Res. 2000, 877: 379); and alpha-B crystalline gene encodingalpha-B crystalline protein involved in protein aggregation diseases,including cardiomyopathy (Cell 2007, 130: 427); alpha1-antitrypsin geneencoding alpha1-antitrypsin protein involved in chronic obstructivepulmonary disease (COPD), liver disease and hepatocellular carcinoma(New Engl J. Med. 2002, 346: 45); Ltk gene encoding leukocyte tyrosinekinase protein involved in systemic lupus erythematosus (Hum. Mol. Gen.2004, 13: 171); PCSK9 gene encoding PCSK9 protein involved inhypercholesterolemia (Hum Mutat. 2009, 30: 520); prolactin receptor geneencoding prolactin receptor protein involved in breast tumors (Proc.Natl. Assoc. Sci. 2008, 105: 4533); CCL5 gene encoding the chemokineCCL5 involved in COPD and asthma (Eur. Respir. J. 2008, 32: 327); PTPN22gene encoding PTPN22 protein involved in Type 1 diabetes, Rheumatoidarthritis, Graves disease, and SLE (Proc. Natl. Assoc. Sci. 2007, 104:19767); androgen receptor gene encoding the androgen receptor proteininvolved in spinal and bulbar muscular atrophy or Kennedy's disease (JSteroid Biochem. Mol. Biol. 2008, 108: 245); CHMP4B gene encodingchromatin modifying protein-4B involved in progressive childhoodposterior subcapsular cataracts (Am. J. Hum. Genet. 2007, 81: 596);FXR/NR1H4 gene encoding Farnesoid X receptor protein involved incholesterol gallstone disease, arthrosclerosis and diabetes (Mol.Endocrinol. 2007, 21: 1769); ABCA1 gene encoding ABCA1 protein involvedin cardiovascular disease (Transl. Res. 2007, 149: 205); CaSR geneencoding the calcium sensing receptor protein involved in primaryhypercalciuria (Kidney Int. 2007, 71: 1155); alpha-globin gene encodingalpha-globin protein involved in alpha-thallasemia (Science 2006, 312:1215); httlpr gene encoding HTTLPR protein involved in obsessivecompulsive disorder (Am. J. Hum. Genet. 2006, 78: 815); AVP geneencoding arginine vasopressin protein in stress-related disorders suchas anxiety disorders and comorbid depression (CNS Neurol. Disord. DrugTargets 2006, 5: 167); GNAS gene encoding G proteins involved incongenital visual defects, hypertension, metabolic syndrome (TrendsPharmacol. Sci. 2006, 27: 260); APAF1 gene encoding APAF1 proteininvolved in a predisposition to major depression (Mol. Psychiatry. 2006,11: 76); TGF-beta1 gene encoding TGF-beta1 protein involved in breastcancer and prostate cancer (Cancer Epidemiol. Biomarkers Prev. 2004, 13:759); AChR gene encoding acetylcholine receptor involved in congentialmyasthenic syndrome (Neurology 2004, 62: 1090); P2Y12 gene encodingadenosine diphosphate (ADP) receptor protein involved in risk ofperipheral arterial disease (Circulation 2003, 108: 2971); LQT1 geneencoding LQT1 protein involved in atrial fibrillation (Cardiology 2003,100: 109); RET protooncogene encoding RET protein involved in sporadicpheochromocytoma (J. Clin. Endocrinol. Metab. 2003, 88: 4911); filamin Agene encoding filamin A protein involved in various congenitalmalformations (Nat. Genet. 2003, 33: 487); TARDBP gene encoding TDP-43protein involved in amyotrophic lateral sclerosis (Hum. Mol. Gene.t2010, 19: 671); SCA3 gene encoding ataxin-3 protein involved inMachado-Joseph disease (PLoS One 2008, 3: e3341); SCAT gene encodingataxin-7 protein involved in spino-cerebellar ataxia-7 (PLoS One 2009,4: e7232); and HTT gene encoding huntingtin protein involved inHuntington's disease (Neurobiol Dis. 1996, 3:183); and the CA4 geneencoding carbonic anhydrase 4 protein, CRX gene encoding cone-rodhomeobox transcription factor protein, FSCN2 gene encoding retinalfascin homolog 2 protein, IMPDH1 gene encoding inosine monophosphatedehydrogenase 1 protein, NR2E3 gene encoding nuclear receptor subfamily2 group E3 protein, NRL gene encoding neural retina leucine zipperprotein, PRPF3 (RP18) gene encoding pre-mRNA splicing factor 3 protein,PRPF8 (RP13) gene encoding pre-mRNA splicing factor 8 protein, PRPF31(RP11) gene encoding pre-mRNA splicing factor 31 protein, RDS geneencoding peripherin 2 protein, ROM1 gene encoding rod outer membraneprotein 1 protein, RHO gene encoding rhodopsin protein, RP1 geneencoding RP1 protein, RPGR gene encoding retinitis pigmentosa GTPaseregulator protein, all of which are involved in Autosomal DominantRetinitis Pigmentosa disease (Adv Exp Med. Biol. 2008, 613:203)

In certain embodiments, the mutant allele is associated with any diseasefrom the group consisting of Alzheimer's disease, Creutzfeldt-Jakobdisease, fatal familial insomnia, Alexander disease, Parkinson'sdisease, amyotrophic lateral sclerosis, dentato-rubral andpallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia,cardiomyopathy, chronic obstructive pulmonary disease (COPD), liverdisease, hepatocellular carcinoma, systemic lupus erythematosus,hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoidarthritis, Graves disease, SLE, spinal and bulbar muscular atrophy,Kennedy's disease, progressive childhood posterior subcapsularcataracts, cholesterol gallstone disease, arthrosclerosis,cardiovascular disease, primary hypercalciuria, alpha-thallasemia,obsessive compulsive disorder, Anxiety, comorbid depression, congenitalvisual defects, hypertension, metabolic syndrome, prostate cancer,congential myasthenic syndrome, peripheral arterial disease, atrialfibrillation, sporadic pheochromocytoma, congenital malformations,Machado-Joseph disease, Huntington's disease, and Autosomal DominantRetinitis Pigmentosa disease.

i. Certain Huntingtin Targets

In certain embodiments, an allelic variant of huntingtin is selectivelyreduced. Nucleotide sequences that encode huntingtin include, withoutlimitation, the following: GENBANK Accession No. NT_(—)006081.18,truncated from nucleotides 1566000 to 1768000 (replaced by GENBANKAccession No. NT_(—)006051), incorporated herein as SEQ ID NO: 1, andNM_(—)002111.6, incorporated herein as SEQ ID NO: 2.

Table 14 provides SNPs found in the GM04022, GM04281, GM02171, andGM02173B cell lines. Also provided are the allelic variants found ateach SNP position, the genotype for each of the cell lines, and thepercentage of HD patients having a particular allelic variant. Forexample, the two allelic variants for SNP rs6446723 are T and C. TheGM04022 cell line is heterozygous TC, the GM02171 cell line ishomozygous CC, the GM02173 cell line is heterozygous TC, and the GM04281cell line is homozygous TT. Fifty percent of HD patients have a T at SNPposition rs6446723.

TABLE 14 Allelic Variations for SNPs Associated with HD SNP VariationGM04022 GM02171 GM02173 GM04281 TargetPOP allele rs6446723 T/C TC CC TCTT 0.50 T rs3856973 A/G AG AA AG GG 0.50 G rs2285086 A/G AG GG AG AA0.50 A rs363092 A/C AC AA AC CC 0.49 C rs916171 C/G GC GG GC CC 0.49 Crs6844859 T/C TC CC TC TT 0.49 T rs7691627 A/G AG AA AG GG 0.49 Grs4690073 A/G AG AA AG GG 0.49 G rs2024115 A/G AG GG AG AA 0.48 Ars11731237 T/C CC CC TC TT 0.43 T rs362296 A/C CC AC AC AC 0.42 Crs10015979 A/G AA AA AG GG 0.42 G rs7659144 C/G CG CG CG CC 0.41 Crs363096 T/C CC CC TC TT 0.40 T rs362273 A/G AA AG AG AA 0.39 Ars16843804 T/C CC TC TC CC 0.38 C rs362271 A/G GG AG AG GG 0.38 Grs362275 T/C CC TC TC CC 0.38 C rs3121419 T/C CC TC TC CC 0.38 Crs362272 A/G GG — AG GG 0.38 G rs3775061 A/G AA AG AG AA 0.38 Ars34315806 T/C CC TC TC CC 0.38 C rs363099 T/C CC TC TC CC 0.38 Crs2298967 T/C TT TC TC TT 0.38 T rs363088 A/T AA TA TA AA 0.38 Ars363064 T/C CC TC TC CC 0.35 C rs363102 A/G AG AA AA AA 0.23 Grs2798235 A/G AG GG GG GG 0.21 A rs363080 T/C TC CC CC CC 0.21 Trs363072 A/T TA TA AA AA 0.13 A rs363125 A/C AC AC CC CC 0.12 C rs362303T/C TC TC CC CC 0.12 C rs362310 T/C TC TC CC CC 0.12 C rs10488840 A/G AGAG GG GG 0.12 G rs362325 T/C TC TC TT TT 0.11 T rs35892913 A/G GG GG GGGG 0.10 A rs363102 A/G AG AA AA AA 0.09 A rs363096 T/C CC CC TC TT 0.09C rs11731237 T/C CC CC TC TT 0.09 C rs10015979 A/G AA AA AG GG 0.08 Ars363080 T/C TC CC CC CC 0.07 C rs2798235 A/G AG GG GG GG 0.07 Grs1936032 C/G GC CC CC CC 0.06 C rs2276881 A/G GG GG GG GG 0.06 Grs363070 A/G AA AA AA AA 0.06 A rs35892913 A/G GG GG GG GG 0.04 Grs12502045 T/C CC CC CC CC 0.04 C rs6446723 T/C TC CC TC TT 0.04 Crs7685686 A/G AG GG AG AA 0.04 G rs3733217 T/C CC CC CC CC 0.03 Crs6844859 T/C TC CC TC TT 0.03 C rs362331 T/C TC CC TC TT 0.03 C

F. CERTAIN INDICATIONS

In certain embodiments, provided herein are methods of treating ananimal or individual comprising administering one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualor animal has Huntington's disease.

In certain embodiments, compounds targeted to huntingtin as describedherein may be administered to reduce the severity of physiologicalsymptoms of Huntington's disease. In certain embodiments, compoundstargeted to huntingtin as described herein may be administered to reducethe rate of degeneration in an individual or an animal havingHuntington's disease. In certain embodiments, compounds targeted tohuntingtin as described herein may be administered regeneration functionin an individual or an animal having Huntington's disease. In certainembodiments, symptoms of Huntingtin's disease may be reversed bytreatment with a compound as described herein.

In certain embodiments, compounds targeted to huntingtin as describedherein may be administered to ameliorate one or more symptoms ofHuntington's disease. In certain embodiments administration of compoundstargeted to huntingtin as described herein may improve the symptoms ofHuntington's disease as measured by any metric known to those havingskill in the art. In certain embodiments, administration of compoundstargeted to huntingtin as described herein may improve a rodent'srotaraod assay performance. In certain embodiments, administration ofcompounds targeted to huntingtin as described herein may improve arodent's plus maze assay. In certain embodiments, administration ofcompounds targeted to huntingtin as described herein may improve arodent's open field assay performance.

Accordingly, provided herein are methods for ameliorating a symptomassociated with Huntington's disease in a subject in need thereof. Incertain embodiments, provided is a method for reducing the rate of onsetof a symptom associated with Huntington's disease. In certainembodiments, provided is a method for reducing the severity of a symptomassociated with Huntington's disease. In certain embodiments, providedis a method for regenerating neurological function as shown by animprovement of a symptom associated with Huntington's disease. In suchembodiments, the methods comprise administering to an individual oranimal in need thereof a therapeutically effective amount of a compoundtargeted to a huntingtin nucleic acid.

Huntington's disease is characterized by numerous physical,neurological, psychiatric, and/or peripheral symptoms. Any symptom knownto one of skill in the art to be associated with Huntington's diseasecan be ameliorated or otherwise modulated as set forth above in themethods described above. In certain embodiments, the symptom is aphysical symptom selected from the group consisting of restlessness,lack of coordination, unintentionally initiated motions, unintentionallyuncompleted motions, unsteady gait, chorea, rigidity, writhing motions,abnormal posturing, instability, abnormal facial expressions, difficultychewing, difficulty swallowing, difficulty speaking, seizure, and sleepdisturbances. In certain embodiments, the symptom is a cognitive symptomselected from the group consisting of impaired planning, impairedflexibility, impaired abstract thinking, impaired rule acquisition,impaired initiation of appropriate actions, impaired inhibition ofinappropriate actions, impaired short-term memory, impaired long-termmemory, paranoia, disorientation, confusion, hallucination and dementia.In certain embodiments, the symptom is a psychiatric symptom selectedfrom the group consisting of anxiety, depression, blunted affect,egocentrisms, aggression, compulsive behavior, irritability and suicidalideation. In certain embodiments, the symptom is a peripheral symptomselected from the group consisting of reduced brain mass, muscleatrophy, cardiac failure, impaired glucose tolerance, weight loss,osteoporosis, and testicular atrophy.

In certain embodiments, the symptom is restlessness. In certainembodiments, the symptom is lack of coordination. In certainembodiments, the symptom is unintentionally initiated motions. Incertain embodiments, the symptom is unintentionally uncompleted motions.In certain embodiments, the symptom is unsteady gait. In certainembodiments, the symptom is chorea. In certain embodiments, the symptomis rigidity. In certain embodiments, the symptom is writhing motions. Incertain embodiments, the symptom is abnormal posturing. In certainembodiments, the symptom is instability. In certain embodiments, thesymptom is abnormal facial expressions. In certain embodiments, thesymptom is difficulty chewing. In certain embodiments, the symptom isdifficulty swallowing. In certain embodiments, the symptom is difficultyspeaking. In certain embodiments, the symptom is seizures. In certainembodiments, the symptom is sleep disturbances.

In certain embodiments, the symptom is impaired planning. In certainembodiments, the symptom is impaired flexibility. In certainembodiments, the symptom is impaired abstract thinking. In certainembodiments, the symptom is impaired rule acquisition. In certainembodiments, the symptom is impaired initiation of appropriate actions.In certain embodiments, the symptom is impaired inhibition ofinappropriate actions. In certain embodiments, the symptom is impairedshort-term memory. In certain embodiments, the symptom is impairedlong-term memory. In certain embodiments, the symptom is paranoia. Incertain embodiments, the symptom is disorientation. In certainembodiments, the symptom is confusion. In certain embodiments, thesymptom is hallucination. In certain embodiments, the symptom isdementia.

In certain embodiments, the symptom is anxiety. In certain embodiments,the symptom is depression. In certain embodiments, the symptom isblunted affect. In certain embodiments, the symptom is egocentrism. Incertain embodiments, the symptom is aggression. In certain embodiments,the symptom is compulsive behavior. In certain embodiments, the symptomis irritability. In certain embodiments, the symptom is suicidalideation.

In certain embodiments, the symptom is reduced brain mass. In certainembodiments, the symptom is muscle atrophy. In certain embodiments, thesymptom is cardiac failure. In certain embodiments, the symptom isimpaired glucose tolerance. In certain embodiments, the symptom isweight loss. In certain embodiments, the symptom is osteoporosis. Incertain embodiments, the symptom is testicular atrophy.

In certain embodiments, symptoms of Huntington's disease may bequantifiable. For example, osteoporosis may be measured and quantifiedby, for example, bone density scans. For such symptoms, in certainembodiments, the symptom may be reduced by about 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined byany two of these values.

In certain embodiments, provided are methods of treating an individualcomprising administering one or more pharmaceutical compositions asdescribed herein. In certain embodiments, the individual hasHuntington's disease.

In certain embodiments, administration of an antisense compound targetedto a huntingtin nucleic acid results in reduction of huntingtinexpression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to huntingtin are used for the preparationof a medicament for treating a patient suffering or susceptible toHuntington's disease.

G. CERTAIN PHARMACEUTICAL COMPOSITIONS

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising one or more antisense compound. In certainembodiments, such pharmaceutical composition comprises a suitablepharmaceutically acceptable diluent or carrier. In certain embodiments,a pharmaceutical composition comprises a sterile saline solution and oneor more antisense compound. In certain embodiments, such pharmaceuticalcomposition consists of a sterile saline solution and one or moreantisense compound. In certain embodiments, the sterile saline ispharmaceutical grade saline. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound and sterile water.In certain embodiments, a pharmaceutical composition consists of one ormore antisense compound and sterile water. In certain embodiments, thesterile saline is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active antisense oligomericcompound.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain.

H. ADMINISTRATION

In certain embodiments, the compounds and compositions as describedherein are administered parenterally.

In certain embodiments, parenteral administration is by infusion.Infusion can be chronic or continuous or short or intermittent. Incertain embodiments, infused pharmaceutical agents are delivered with apump. In certain embodiments, parenteral administration is by injection.

In certain embodiments, compounds and compositions are delivered to theCNS. In certain embodiments, compounds and compositions are delivered tothe cerebrospinal fluid. In certain embodiments, compounds andcompositions are administered to the brain parenchyma. In certainembodiments, compounds and compositions are delivered to an animal byintrathecal administration, or intracerebroventricular administration.Broad distribution of compounds and compositions, described herein,within the central nervous system may be achieved with intraparenchymaladministration, intrathecal administration, or intracerebroventricularadministration.

In certain embodiments, parenteral administration is by injection. Theinjection may be delivered with a syringe or a pump. In certainembodiments, the injection is a bolus injection. In certain embodiments,the injection is administered directly to a tissue, such as striatum,caudate, cortex, hippocampus and cerebellum.

Therefore, in certain embodiments, delivery of a compound or compositiondescribed herein can affect the pharmacokinetic profile of the compoundor composition. In certain embodiments, injection of a compound orcomposition described herein, to a targeted tissue improves thepharmacokinetic profile of the compound or composition as compared toinfusion of the compound or composition. In a certain embodiment, theinjection of a compound or composition improves potency compared tobroad diffusion, requiring less of the compound or composition toachieve similar pharmacology. In certain embodiments, similarpharmacology refers to the amount of time that a target mRNA and/ortarget protein is down-regulated (e.g. duration of action). In certainembodiments, methods of specifically localizing a pharmaceutical agent,such as by bolus injection, decreases median effective concentration(EC50) by a factor of about 50 (e.g. 50 fold less concentration intissue is required to achieve the same or similar pharmacodynamiceffect). In certain embodiments, methods of specifically localizing apharmaceutical agent, such as by bolus injection, decreases medianeffective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or50. In certain embodiments the pharmaceutical agent in an antisensecompound as further described herein. In certain embodiments, thetargeted tissue is brain tissue. In certain embodiments the targetedtissue is striatal tissue. In certain embodiments, decreasing EC50 isdesirable because it reduces the dose required to achieve apharmacological result in a patient in need thereof.

In certain embodiments, an antisense oligonucleotide is delivered byinjection or infusion once every month, every two months, every 90 days,every 3 months, every 6 months, twice a year or once a year.

I. CERTAIN COMBINATION THERAPIES

In certain embodiments, one or more pharmaceutical compositions areco-administered with one or more other pharmaceutical agents. In certainembodiments, such one or more other pharmaceutical agents are designedto treat the same disease, disorder, or condition as the one or morepharmaceutical compositions described herein. In certain embodiments,such one or more other pharmaceutical agents are designed to treat adifferent disease, disorder, or condition as the one or morepharmaceutical compositions described herein. In certain embodiments,such one or more other pharmaceutical agents are designed to treat anundesired side effect of one or more pharmaceutical compositions asdescribed herein. In certain embodiments, one or more pharmaceuticalcompositions are co-administered with another pharmaceutical agent totreat an undesired effect of that other pharmaceutical agent. In certainembodiments, one or more pharmaceutical compositions are co-administeredwith another pharmaceutical agent to produce a combinational effect. Incertain embodiments, one or more pharmaceutical compositions areco-administered with another pharmaceutical agent to produce asynergistic effect.

In certain embodiments, one or more pharmaceutical compositions and oneor more other pharmaceutical agents are administered at the same time.In certain embodiments, one or more pharmaceutical compositions and oneor more other pharmaceutical agents are administered at different times.In certain embodiments, one or more pharmaceutical compositions and oneor more other pharmaceutical agents are prepared together in a singleformulation. In certain embodiments, one or more pharmaceuticalcompositions and one or more other pharmaceutical agents are preparedseparately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of includeantipsychotic agents, such as, e.g., haloperidol, chlorpromazine,clozapine, quetiapine, and olanzapine; antidepressant agents, such as,e.g., fluoxetine, sertraline hydrochloride, venlafaxine andnortriptyline; tranquilizing agents such as, e.g., benzodiazepines,clonazepam, paroxetine, venlafaxin, and beta-blockers; mood-stabilizingagents such as, e.g., lithium, valproate, lamotrigine, andcarbamazepine; paralytic agents such as, e.g., Botulinum toxin; and/orother experimental agents including, but not limited to, tetrabenazine(Xenazine), creatine, coenzyme Q10, trehalose, docosahexanoic acids,ACR16, ethyl-EPA, atomoxetine, citalopram, dimebon, memantine, sodiumphenylbutyrate, ramelteon, ursodiol, zyprexa, xenasine, tiapride,riluzole, amantadine, [123I]MNI-420, atomoxetine, tetrabenazine,digoxin, detromethorphan, warfarin, alprozam, ketoconazole, omeprazole,and minocycline.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligomeric compound having the nucleobase sequence “ATCGATCG”encompasses any oligomeric compounds having such nucleobase sequence,whether modified or unmodified, including, but not limited to, suchcompounds comprising RNA bases, such as those having sequence “AUCGAUCG”and those having some DNA bases and some RNA bases such as “AUCGATCG”and oligomeric compounds having other modified or naturally occurringbases, such as “AT^(me)CGAUCG,” wherein ^(me)C indicates a cytosine basecomprising a methyl group at the 5-position.

EXAMPLES

The following examples illustrate certain embodiments of the presentinvention and are not limiting. Moreover, where specific embodiments areprovided, the inventors have contemplated generic application of thosespecific embodiments. For example, disclosure of an oligonucleotidehaving a particular motif provides reasonable support for additionaloligonucleotides having the same or similar motif. And, for example,where a particular high-affinity modification appears at a particularposition, other high-affinity modifications at the same position areconsidered suitable, unless otherwise indicated.

To allow assessment of the relative effects of nucleobase sequence andchemical modification, throughout the examples, oligomeric compounds areassigned a “Sequence Code.” Oligomeric compounds having the sameSequence Code have the same nucleobase sequence. Oligomeric compoundshaving different Sequence Codes have different nucleobase sequences.

Example 1 Modified Antisense Oligonucleotides Targeting Human Target-X

Antisense oligonucleotides were designed targeting a Target-X nucleicacid and were tested for their effects on Target-X mRNA in vitro. ISIS407939, which was described in an earlier publication (WO 2009/061851)was also tested.

The newly designed chimeric antisense oligonucleotides and their motifsare described in Table 15. The internucleoside linkages throughout eachgapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d”indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate6′-(S)—CH₃ bicyclic nucleoside (e.g cEt) nucleosides. Nucleosidesfollowed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides. “N”indicates modified or naturally occurring nucleobases (A, T, C, G, U, or5-methyl C).

Each gapmer listed in Table 15 is targeted to the human Target-X genomicsequence.

Activity of the newly designed gapmers was compared to a 5-10-5 2′-MOEgapmer, ISIS 407939 targeting human Target-X and is further described inUSPN XXX, incorporated herein by reference. Cultured Hep3B cells at adensity of 20,000 cells per well were transfected using electroporationwith 2,000 nM antisense oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and Target-XmRNA levels were measured by quantitative real-time PCR. Human primerprobe set RTS2927 was used to measure mRNA levels. Target-X mRNA levelswere adjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of Target-X, relative tountreated control cells, and indicate that several of the newly designedantisense oligonucleotides are more potent than ISIS 407939. A total of771 oligonucleotides were tested. Only those oligonucleotides which wereselected for further studies are shown in Table 15. Each of the newlydesigned antisense oligonucleotides provided in Table 1 achieved greaterthan 80% inhibition and, therefore, are more active than ISIS 407939.

TABLE 15 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X Wing SEQ ISIS % Gap Chemistry SEQID Sequence (5′ to 3′) NO inhibition Motif Chemistry 5′ 3′ CODE NONkNkNkNdNdNdNdNkNd 473359  92 3-10-3 Deoxy/ kkk eee 21 6 NdNdNdNdNeNeNecEt NkNkNkNdNdNdNdNkNd 473360  96 3-10-3 Deoxy/ kkk eee 22 6NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNdNd 473168  94 3-10-3 Full kkk kkk 236 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473317  95 3-10-3 Full kkk eee23 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473471  90 3-10-3 Deoxy/kkk eee 23 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473620  94 5-9-2 Fullkdkdk ee 23 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 473019  88 2-10-2Full kk kk 24 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 473020  93 2-10-2Full kk kk 25 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473321  93 3-10-3Full kkk eee 26 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNdNd 473322  943-10-3 Full kkk eee 27 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNdNd 473323 96 3-10-3 Full kkk eee 28 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNdNd473326  94 3-10-3 Full kkk eee 29 6 NdNdNdNdNeNeNe deoxyNkNkNkNdNdNdNdNkNd 473480  92 3-10-3 Deoxy/ kkk eee 29 6 NdNdNdNdNeNeNecEt NkNkNkNdNdNdNdNdNd 473178  96 3-10-3 Full kkk kkk 30 6NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473327  96 3-10-3 Full kkk eee30 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473481  93 3-10-3 Deoxy/kkk eee 30 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473630  89 5-9-2 Fullkdkdk ee 30 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 473029  96 2-10-2Full kk kk 31 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 472925  93 2-10-2Full kk kk 32 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 472926  85 2-10-2Full kk kk 33 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473195  97 3-10-3Full kkk kkk 34 6 NdNdNdNdNkNkNk deoxy NkNkNdNdNdNdNdNdNd 473046  902-10-2 Full kk kk 35 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 472935  922-10-2 Full kk kk 36 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473089  953-10-3 Full kkk kkk 37 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473350 93 3-10-3 Full kkk eee 38 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNdNd473353  93 3-10-3 Full kkk eee 39 6 NdNdNdNdNeNeNe deoxyNkNkNdNdNdNdNdNdNd 473055  91 2-10-2 Full kk kk 40 7 NdNdNdNkNk deoxyNkNkNkNdNdNdNdNkNd 473392  95 3-10-3 Deoxy/ kkk eee 41 6 NdNdNdNdNeNeNecEt NkNkNkNdNdNdNdNdNd 473095 100 3-10-3 Full kkk kkk 42 6NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473244  99 3-10-3 Full kkk eee42 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473393  99 3-10-3 Deoxy/kkk eee 42 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473547  98 5-9-2 Fullkdkdk ee 42 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 472942  87 2-10-2Full kk kk 43 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473098  97 3-10-3Full kkk kkk 44 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNkNd 473408  923-10-3 Deoxy/ kkk eee 45 6 NdNdNdNdNeNeNe cEt NkNkNdNdNdNdNdNdNd 472958 89 2-10-2 Full kk kk 46 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 472959 90 2-10-2 Full kk kk 47 7 NdNdNdNkNk deoxy NkNdNkNdNkNdNdNdNd 473566 94 5-9-2 Full kdkdk ee 48 6 NdNdNdNdNdNeNe deoxy NkNdNkNdNkNdNdNdNd473567  95 5-9-2 Full kdkdk ee 49 6 NdNdNdNdNdNeNe deoxyNkNdNkNdNkNdNdNdNd 473569  92 5-9-2 Full kdkdk ee 50 6 NdNdNdNdNdNeNedeoxy NkNkNdNdNdNdNdNdNd 457851  90 2-10-2 Full kk kk 51 7 NdNdNdNkNkdeoxy NkNkNdNdNdNdNdNdNd 472970  91 2-10-2 Full kk kk 32 7 NdNdNdNkNkdeoxy NkNkNkNdNdNdNdNdNd 473125  90 3-10-3 Full kkk kkk 53 6NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473274  98 3-10-3 Full kkk eee53 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473428  90 3-10-3 Deoxy/kkk eee 53 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473577  93 5-9-2 Fullkdkdk ee 53 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 472976  97 2-10-2Full kk kk 54 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNd 472983  94 2-10-2 Fullkk kk 55 7 NdNdNdNdNkNk deoxy NkNkNdNdNdNdNdNd 472984  90 2-10-2 Full kkkk 56 7 NdNdNdNdNkNk deoxy NkNkNkNdNdNdNdNd 473135  97 3-10-3 Full kkkkkk 57 6 NdNdNdNdNdNkNkNk deoxy NkNkNdNdNdNdNdNd 472986  95 2-10-2 Fullkk kk 58 7 NdNdNdNdNkNk deoxy NkNkNkNdNdNdNdNd 473137  95 3-10-3 Fullkkk kkk 59 6 NdNdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNd 473286  95 3-10-3Full kkk eee 59 6 NdNdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473440  883-10-3 Deoxy/ kkk eee 59 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNd 473589 97 5-9-2 Full kdkdk ee 59 6 NdNdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNd472988  85 2-10-2 Full kk kk 60 7 NdNdNdNdNkNk deoxy NkNkNkNdNdNdNdNd473140  96 3-10-3 Full kkk kkk 61 6 NdNdNdNdNdNkNkNk deoxyNkNkNdNdNdNdNdNd 472991  90 2-10-2 Full kk kk 62 7 NdNdNdNdNkNk deoxyNkNkNkNdNdNdNdNkNd 473444  94 3-10-3 Deoxy/ kkk eee 63 6 NdNdNdNdNeNeNecEt NkNkNkNdNdNdNdNd 473142  96 3-10-3 Full kkk kkk 64 6NdNdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNd 473291  95 3-10-3 Full kkk eee64 6 NdNdNdNdNdNeNeNe deoxy NkNdNkNdNkNdNdNd 473594  95 5-9-2 Full kdkdkee 64 6 NdNdNdNdNdNdNeNe deoxy NkNkNkNdNdNdNdNdNd 473143  97 3-10-3 Fullkkk kkk 65 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNd 473292  96 3-10-3Full kkk eee 65 6 NdNdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473446  963-10-3 Deoxy/ kkk eee 65 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473595 84 5-9-2 Full kdkdk ee 65 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd472994  96 2-10-2 Full kk kk 66 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd473144  98 3-10-3 Full kkk kkk 67 6 NdNdNdNdNkNkNk deoxyNkNkNkNdNdNdNdNdNd 473293  96 3-10-3 Full kkk eee 67 6 NdNdNdNdNeNeNedeoxy NkNkNdNdNdNdNdNdNd 472995  96 2-10-2 Full kk kk 68 7 NdNdNdNkNkdeoxy NkNkNkNdNdNdNdNd 473294  91 3-10-3 Full kkk eee 69 6NdNdNdNdNdNeNeNe deoxy NkNdNkNdNkNdNdNdNd 473597  94 5-9-2 Full kdkdk ee69 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 472996  94 2-10-2 Full kkkk 70 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNd 473295  92 3-10-3 Full kkk eee71 6 NdNdNdNdNdNeNeNe deoxy NeNeNeNeNeNdNdNdNdNd 407939  80 5-10-5 Fulleeeee eeee 72 8 NdNdNdNdNdNeNeNeNeNe deoxy e NkNkNkNdNdNdNdNdNd 473296 98 3-10-3 Full kkk eee 73 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd473450  95 3-10-3 Deoxy/ kkk eee 73 6 NdNdNdNdNeNeNe cEtNkNkNdNdNdNdNdNdNd 472998  97 2-10-2 Full kk kk 74 7 NdNdNdNkNk deoxy e= 2′MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 2 Modified Antisense Oligonucleotides Comprising 6′-(S)—CH₃Bicyclic Nucleoside (cEt) and F-HNA Modifications Targeting HumanTarget-X

Additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid and were tested for their effects on Target-X mRNA invitro. ISIS 407939 was also tested.

The chimeric antisense oligonucleotides and their motifs are describedin Table 16. The internucleoside linkages throughout each gapmer arephosphorothioate linkages (P═S). Nucleosides followed by “d” indicate2′-deoxyribonucleosides. Nucleosides followed by “k” indicate 6′-(S)—CH₃bicyclic nucleosides (e.g cEt). Nucleosides followed by “e” indicate2′-O-methoxyethyl (2′-MOE) modified nucleosides. Nucleosides followed by‘g’ indicate F-HNA modified nucleosides. “N” indicates modified ornaturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

Each gapmer listed in Table 16 is targeted to the human Target-X genomicsequence.

Activity of the newly designed gapmers was compared to a 5-10-5 2′-MOEgapmer, ISIS 407939 targeting human Target-X. Cultured Hep3B cells at adensity of 20,000 cells per well were transfected using electroporationwith 2,000 nM antisense oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and Target-XmRNA levels were measured by quantitative real-time PCR. Human primerprobe set RTS2927 was used to measure mRNA levels. Target-X mRNA levelswere adjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of Target-X, relative tountreated control cells, and demonstrate that several of the newlydesigned gapmers are more potent than ISIS 407939. A total of 765oligonucleotides were tested. Only those oligonucleotides which wereselected for further studies are shown in Table 16. All but one of thenewly designed antisense oligonucleotides provided in Table 16 achievedgreater than 30% inhibition and, therefore, are more active than ISIS407939.

TABLE 16 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X Wing SEQ ISIS % Gap Chemistry SEQID Sequence (5′ to 3′) No inhibition Motif Chemistry 5′ 3′ CODE NONgNgNdNdNdNdNdNdNd 482838 81 2-10-2 Full gg gg 25 7 NdNdNdNgNg deoxyNgNgNgNdNdNdNdNdNd 482992 93 3-10-3 Full ggg ggg 28 6 NdNdNdNdNgNgNgdeoxy NgNgNgNdNdNdNdNdNd 482996 97 3-10-3 Full ggg ggg 30 6NdNdNdNdNgNgNg deoxy NgNdNgNdNgNdNdNdNd 483284 82 5-9-2 Full gdgdg ee 236 NdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNdNd 483289 70 5-9-2 Full gdgdg ee27 6 NdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNdNd 483290 80 5-9-2 Full gdgdgee 28 6 NdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNdNd 483294 69 5-9-2 Fullgdgdg ee 30 6 NdNdNdNdNdNeNe deoxy NgNgNdNdNdNdNdNdNd 483438 81 2-10-4Full gg eeee 23 6 NdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNd 483444 842-10-4 Full gg eeee 28 6 NdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNd 48344877 2-10-4 Full gg eeee 30 6 NdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNd482847 79 2-10-2 Full gg gg 31 7 NdNdNdNgNg deoxy NgNgNdNdNdNdNdNdNd482747 85 2-10-2 Full gg gg 32 7 NdNdNdNgNg deoxy NgNgNdNdNdNdNdNdNd482873 81 2-10-2 Full gg gg 40 7 NdNdNdNgNg deoxy NgNgNdNdNdNdNdNdNdNd482874 82 2-10-2 Full gg gg 75 7 NdNdNgNg deoxy NgNgNdNdNdNdNdNd 48287582 2-10-2 Full gg gg 76 7 NdNdNdNdNgNg deoxy NgNgNgNdNdNdNdNd 482896 953-10-3 Full ggg ggg 77 6 NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNdNd483019 89 3-10-3 Full ggg ggg 38 6 NdNdNdNdNgNgNg deoxyNgNdNgNdNdNdNdNdNd 483045 92 3-10-3 Full gdg gdg 77 6 NdNdNdNdNgNdNgdeoxy NgNdNgNdNgNdNdNdNd 483194 64 3-10-3 Full gdg gdg 77 6NdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNdNd 483317 79 5-9-2 Full gdgdg ee 386 NdNdNdNdNdNeNe deoxy NgNgNdNdNdNdNdNdNd 483343 75 2-10-4 Full gg eeee57 6 NdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNdNdN 483471 76 2-10-4 Full ggeeee 38 6 dNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNd 483478 20 2-10-4 Full ggeeee 78 6 NdNdNdNeNeNeNe deoxy NeNeNeNeNeNdNdNdNdNd 407939 30 5-10-5Full eeeee eeeee 72 8 NdNdNdNdNdNeNeNeNeNe deoxy NgNgNdNdNdNdNdNd 48278483 2-10-2 Full gg gg 79 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482794 912-10-2 Full gg gg 54 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482804 802-10-2 Full gg gg 58 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482812 812-10-2 Full gg gg 66 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482813 922-10-2 Full gg gg 68 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482814 942-10-2 Full gg gg 70 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482815 812-10-2 Full gg gg 80 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482816 712-10-2 Full gg gg 74 7 NdNdNdNdNgNg deoxy NgNgNgNdNdNdNdNd 482916 903-10-3 Full ggg ggg 44 6 NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNd 48293289 3-10-3 Full ggg ggg 48 6 NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNd482953 93 3-10-3 Full ggg ggg 57 6 NdNdNdNdNdNgNgNg deoxyNgNgNgNdNdNdNdNd 482962 97 3-10-3 Full ggg ggg 67 6 NdNdNdNdNdNgNgNgdeoxy NgNgNgNdNdNdNdNd 482963 96 3-10-3 Full ggg ggg 69 6NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNd 482965 89 3-10-3 Full ggg ggg 736 NdNdNdNdNdNgNgNg deoxy NgNdNgNdNdNdNdNd 483065 69 3-10-3 Full ggg ggg44 6 NdNdNdNdNdNgNdNg deoxy NgNdNgNdNdNdNdNd 483092 89 3-10-3 Full gdggdg 53 6 NdNdNdNdNdNgNdNg deoxy NgNdNgNdNgNdNdNd 483241 79 5-9-2 Fullgdgdg ee 53 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd 483253 76 5-9-2Full gdgdg ee 59 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd 483258 705-9-2 Full gdgdg ee 64 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd 48326062 5-9-2 Full gdgdg ee 67 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd483261 76 5-9-2 Full gdgdg ee 69 6 NdNdNdNdNdNdNeNe deoxyNgNdNgNdNgNdNdNd 483262 75 5-9-2 Full gdgdg ee 71 6 NdNdNdNdNdNdNeNedeoxy NgNdNgNdNgNdNdNd 483263 73 5-9-2 Full gdgdg ee 73 6NdNdNdNdNdNdNeNe deoxy NgNgNdNdNdNdNdNd 483364 78 2-10-4 Full gg eeee 816 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483395 86 2-10-4 Full gg eeee53 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483413 83 2-10-4 Full ggeeee 65 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483414 76 2-10-4 Fullgg eeee 67 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483415 85 2-10-4Full gg eeee 69 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483416 772-10-4 Full gg eeee 71 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 48341783 2-10-4 Full gg eeee 73 6 NdNdNdNdNeNeNeNe deoxy e = 2′-MOE, d =2′-deoxyribonucleoside, g = F-HNA

Example 3 Modified Antisense Oligonucleotides Comprising 2′-MOE and6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt) Modifications Targeting HumanTarget-X

Additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid and were tested for their effects on Target-X mRNA invitro. ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS416438, which were described in an earlier publication (WO 2009/061851)were also tested.

The newly designed chimeric antisense oligonucleotides are 16nucleotides in length and their motifs are described in Table 17. Thechemistry column of Table 17 presents the sugar motif of eacholigonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE)nucleoside, “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g cEt) and“d” indicates a 2′-deoxyribonucleoside. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytosineresidues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 17 is targeted to the human Target-X genomicsequence.

Activity of the newly designed gapmers was compared to ISIS 403052, ISIS407594, ISIS 407606, ISIS 407939, and ISIS 416438. Cultured Hep3B cellsat a density of 20,000 cells per well were transfected usingelectroporation with 2,000 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and Target-X mRNA levels were measured by quantitative real-timePCR. Human primer probe set RTS2927 (described hereinabove in Example 1)was used to measure mRNA levels. Target-X mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN. Results arepresented as percent inhibition of Target-X, relative to untreatedcontrol cells. A total of 380 oligonucleotides were tested. Only thoseoligonucleotides which were selected for further studies are shown inTable 17. Each of the newly designed antisense oligonucleotides providedin Table 17 achieved greater than 64% inhibition and, therefore, aremore potent than each of ISIS 403052, ISIS 407594, ISIS 407606, ISIS407939, and ISIS 416438.

TABLE 17 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X ISIS No Chemistry Motif %inhibition SEQ CODE 403052 eeeee-(d10)-eeeee 5-10-5 64 82 407594eeeee-(d10)-eeeee 5-10-5 40 83 407606 eeeee-(d10)-eeeee 5-10-5 39 84407939 eeeee-(d10)-eeeee 5-10-5 57 72 416438 eeeee-(d10)-eeeee 5-10-5 6285 484487 kdk-(d10)-dkdk 3-10-3 91 77 484539 kdk-d(10)-kdk 3-10-3 92 53484546 kdk-d(10)-kdk 3-10-3 92 86 484547 kdk-d(10)-kdk 3-10-3 89 87484549 kdk-d(10)-kdk 3-10-3 91 57 484557 kdk-d(10)-kdk 3-10-3 92 65484558 kdk-d(10)-kdk 3-10-3 94 67 484559 kdk-d(10)-kdk 3-10-3 90 69484582 kdk-d(10)-kdk 3-10-3 88 23 484632 kk-d(10)-eeee 2-10-4 90 88484641 kk-d(10)-eeee 2-10-4 91 77 484679 kk-d(10)-eeee 2-10-4 90 49484693 kk-d(10)-eeee 2-10-4 93 53 484711 kk-d(10)-eeee 2-10-4 92 65484712 kk-d(10)-eeee 2-10-4 92 67 484713 kk-d(10)-eeee 2-10-4 85 69484714 kk-d(10)-eeee 2-10-4 83 71 484715 kk-d(10)-eeee 2-10-4 93 73484736 kk-d(10)-eeee 2-10-4 89 23 484742 kk-d(10)-eeee 2-10-4 93 28484746 kk-d(10)-eeee 2-10-4 88 30 484771 kk-d(10)-eeee 2-10-4 89 89 e =2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 4 Antisense Inhibition of Human Target-X with 5-10-5 2′-MOEGapmers

Additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid and were tested for their effects on Target-X mRNA invitro. Also tested were ISIS 403094, ISIS 407641, ISIS 407643, ISIS407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939,ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS422142, 5-10-5 2′-MOE gapmers targeting human Target-X, which weredescribed in an earlier publication (WO 2009/061851), incorporatedherein by reference.

The newly designed modified antisense oligonucleotides are 20nucleotides in length and their motifs are described in Tables 18 and19. The chemistry column of Tables 18 and 19 present the sugar motif ofeach oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE)nucleoside and “d” indicates a 2′-deoxyribonucleoside. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine residues throughout each oligonucleotideare 5-methylcytosines.

Each gapmer listed in Table 18 is targeted to the human Target-X genomicsequence.

Activity of the newly designed gapmers was compared to ISIS 403094, ISIS407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935,ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087,ISIS 422140, and ISIS 422142. Cultured Hep3B cells at a density of20,000 cells per well were transfected using electroporation with 2,000nM antisense oligonucleotide. After a treatment period of approximately24 hours, RNA was isolated from the cells and Target-X mRNA levels weremeasured by quantitative real-time PCR. Human primer probe set RTS2927(described hereinabove in Example 1) was used to measure mRNA levels.Target-X mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN. Results are presented as percent inhibition ofTarget-X, relative to untreated control cells. A total of 916oligonucleotides were tested. Only those oligonucleotides which wereselected for further studies are shown in Tables 18 and 19.

TABLE 18 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X ISIS No Chemistry % inhibition SEQCODE 490275 e5-d(10)-e5 35 90 490277 e5-d(10)-e5 73 91 490278e5-d(10)-e5 78 92 490279 e5-d(10)-e5 66 93 490323 e5-d(10)-e5 65 94490368 e5-d(10)-e5 78 95 490396 e5-d(10)-e5 76 96 416507 e5-d(10)-e5 7397 422140 e5-d(10)-e5 59 98 422142 e5-d(10)-e5 73 99 416508 e5-d(10)-e575 100 490424 e5-d(10)-e5 57 101 490803 e5-d(10)-e5 70 102 416446e5-d(10)-e5 73 103 416449 e5-d(10)-e5 33 104 407900 e5-d(10)-e5 66 105490103 e5-d(10)-e5 87 106 416455 e5-d(10)-e5 42 107 407910 e5-d(10)-e525 108 490149 e5-d(10)-e5 82 109 403094 e5-d(10)-e5 60 110 416472e5-d(10)-e5 78 111 407641 e5-d(10)-e5 64 112 416477 e5-d(10)-e5 25 113407643 e5-d(10)-e5 78 114 490196 e5-d(10)-e5 81 115 490197 e5-d(10)-e585 116 490208 e5-d(10)-e5 89 117 490209 e5-d(10)-e5 81 118 422086e5-d(10)-e5 90 119 407935 e5-d(10)-e5 91 120 422087 e5-d(10)-e5 89 121407936 e5-d(10)-e5 80 122 407939 e5-d(10)-e5 67 72 e = 2′-MOE, d =2′-deoxynucleoside

TABLE 19 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides ISIS No Motif % inhibition SEQ CODE 407662 e5-d(10)-e576 123 416446 e5-d(10)-e5 73 103 e = 2′-MOE, d = 2′-deoxynucleoside

Example 5 Modified Chimeric Antisense Oligonucleotides Comprising6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt) Modifications at 5′ and 3′ WingRegions Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid and were tested for their effects on Target-X mRNA invitro. ISIS 407939, which was described in an earlier publication (WO2009/061851) were also tested. ISIS 457851, ISIS 472925, ISIS 472926,ISIS 472935, ISIS 472942, ISIS 472958, ISIS 472959, ISIS 472970, ISIS472976, ISIS 472983, ISIS 472984, ISIS 472988, ISIS 472991, ISIS 472994,ISIS 472995, ISIS 472996, ISIS 472998, and ISIS 473020, described in theExamples above were also included in the screen.

The newly designed chimeric antisense oligonucleotides in Table 20 weredesigned as 2-10-2 cEt gapmers. The newly designed gapmers are 14nucleosides in length, wherein the central gap segment comprises of ten2′-deoxyribonucleosides and is flanked by wing segments on the 5′direction and the 3′ direction comprising five nucleosides each. Eachnucleoside in the 5′ wing segment and each nucleoside in the 3′ wingsegment comprises 6′-(S)—CH₃ bicyclic nucleoside (e.g cEt) modification.The internucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine residues throughout each gapmer are5-methylcytosines.

Each gapmer listed in Table 20 is targeted to the human Target-X genomicsequence.

Activity of the newly designed oligonucleotides was compared to ISIS407939. Cultured Hep3B cells at a density of 20,000 cells per well weretransfected using electroporation with 2,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and Target-X mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS2927 (describedhereinabove in Example 1) was used to measure mRNA levels. Target-X mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN. Results are presented as percent inhibition of Target-X,relative to untreated control cells. A total of 614 oligonucleotideswere tested. Only those oligonucleotides which were selected for furtherstudies are shown in Table 20. Many of the newly designed antisenseoligonucleotides provided in Table 20 achieved greater than 72%inhibition and, therefore, are more potent than ISIS 407939.

TABLE 20 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X ISIS No % inhibition Motif WingChemistry SEQ CODE 407939 72 5-10-5 cEt 72 473020 90 2-10-2 cEt 25492465 83 2-10-2 cEt 124 492467 74 2-10-2 cEt 125 492492 84 2-10-2 cEt126 492494 91 2-10-2 cEt 127 492503 89 2-10-2 cEt 128 492530 91 2-10-2cEt 129 492534 91 2-10-2 cEt 130 492536 90 2-10-2 cEt 131 492541 842-10-2 cEt 132 492545 89 2-10-2 cEt 133 492566 90 2-10-2 cEt 134 49257182 2-10-2 cEt 135 492572 89 2-10-2 cEt 136 492573 90 2-10-2 cEt 137492574 92 2-10-2 cEt 138 492575 88 2-10-2 cEt 139 492593 83 2-10-2 cEt140 492617 91 2-10-2 cEt 141 492618 92 2-10-2 cEt 142 492619 90 2-10-2cEt 143 492621 75 2-10-2 cEt 144 492104 89 2-10-2 cEt 145 492105 862-10-2 cEt 146 492189 88 2-10-2 cEt 147 492194 92 2-10-2 cEt 148 49219590 2-10-2 cEt 149 472925 87 2-10-2 cEt 32 492196 91 2-10-2 cEt 150472926 88 2-10-2 cEt 33 492205 92 2-10-2 cEt 151 492215 77 2-10-2 cEt152 492221 79 2-10-2 cEt 153 472935 82 2-10-2 cEt 36 492234 86 2-10-2cEt 154 472942 85 2-10-2 cEt 43 492276 75 2-10-2 cEt 155 492277 752-10-2 cEt 156 492306 85 2-10-2 cEt 157 492317 93 2-10-2 cEt 158 47295892 2-10-2 cEt 46 472959 88 2-10-2 cEt 47 492329 88 2-10-2 cEt 159 49233195 2-10-2 cEt 160 492333 85 2-10-2 cEt 161 492334 88 2-10-2 cEt 162457851 89 2-10-2 cEt 51 472970 92 2-10-2 cEt 52 492365 69 2-10-2 cEt 163472976 94 2-10-2 cEt 54 472983 76 2-10-2 cEt 55 472984 72 2-10-2 cEt 56492377 70 2-10-2 cEt 164 492380 80 2-10-2 cEt 165 492384 61 2-10-2 cEt166 472988 59 2-10-2 cEt 60 492388 70 2-10-2 cEt 167 492389 70 2-10-2cEt 168 492390 89 2-10-2 cEt 169 492391 80 2-10-2 cEt 170 472991 842-10-2 cEt 62 492398 88 2-10-2 cEt 171 492399 94 2-10-2 cEt 172 49240191 2-10-2 cEt 173 492403 78 2-10-2 cEt 174 472994 95 2-10-2 cEt 66472995 91 2-10-2 cEt 68 492404 84 2-10-2 cEt 175 492405 87 2-10-2 cEt176 472996 85 2-10-2 cEt 70 492406 43 2-10-2 cEt 177 472998 92 2-10-2cEt 74 492440 89 2-10-2 cEt 178

Example 6 Modified Chimeric Antisense Oligonucleotides Comprising6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt) Modifications at 5′ and 3′ WingRegions Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid and were tested for their effects on Target-X mRNA invitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting humanTarget-X, which was described in an earlier publication (WO2009/061851). ISIS 472998 and ISIS 473046, described in the Examplesabove were also included in the screen.

The newly designed chimeric antisense oligonucleotides in Table 21 weredesigned as 2-10-2 cEt gapmers. The newly designed gapmers are 14nucleosides in length, wherein the central gap segment comprises of ten2′-deoxyribonucleosides and is flanked by wing segments on the 5′direction and the 3′ direction comprising five nucleosides each. Eachnucleoside in the 5′ wing segment and each nucleoside in the 3′ wingsegment comprise 6′-(S)—CH₃ bicyclic nucleoside (e.g cEt) modification.The internucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine residues throughout each gapmer are5-methylcytosines.

Each gapmer listed in Table 21 is targeted to the human Target-X genomicsequence.

Activity of the newly designed gapmers was compared to ISIS 407939.Cultured Hep3B cells at a density of 20,000 cells per well weretransfected using electroporation with 2,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and Target-X mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS2927 (describedhereinabove in Example 1) was used to measure mRNA levels. Target-X mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN. Results are presented as percent inhibition of Target-X,relative to untreated control cells. A total of 757 oligonucleotideswere tested. Only those oligonucleotides which were selected for furtherstudies are shown in Table 21. Each of the newly designed antisenseoligonucleotides provided in Table 21 achieved greater than 67%inhibition and, therefore, are more potent than 407939.

TABLE 21 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X ISIS No % inhibition Motif Wingchemistry SEQ CODE 407939 67 5-10-5 cEt 72 492651 77 2-10-2 cEt 179492652 84 2-10-2 cEt 180 492658 87 2-10-2 cEt 181 492725 74 2-10-2 cEt182 492730 78 2-10-2 cEt 183 492731 72 2-10-2 cEt 184 492784 72 2-10-2cEt 185 492816 70 2-10-2 cEt 186 492818 73 2-10-2 cEt 187 492877 832-10-2 cEt 188 492878 79 2-10-2 cEt 189 492913 73 2-10-2 cEt 190 49291482 2-10-2 cEt 191 492928 76 5-10-5 cEt 192 492938 80 2-10-2 cEt 193492991 91 2-10-2 cEt 194 492992 73 2-10-2 cEt 195 493087 81 2-10-2 cEt196 493114 80 2-10-2 cEt 197 493178 86 2-10-2 cEt 198 493179 69 2-10-2cEt 199 493182 79 2-10-2 cEt 200 493195 71 2-10-2 cEt 201 473046 792-10-2 cEt 35 493201 86 2-10-2 cEt 202 493202 76 2-10-2 cEt 203 49325580 2-10-2 cEt 204 493291 84 2-10-2 cEt 205 493292 90 2-10-2 cEt 206493296 82 2-10-2 cEt 207 493298 77 2-10-2 cEt 208 493299 76 5-10-5 cEt209 493304 77 2-10-2 cEt 210 493312 75 2-10-2 cEt 211 493333 76 2-10-2cEt 212 472998 85 2-10-2 cEt 74

Example 7 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3BCells

Antisense oligonucleotides from the studies above, exhibiting in vitroinhibition of Target-X mRNA, were selected and tested at various dosesin Hep3B cells. Cells were plated at a density of 20,000 cells per welland transfected using electroporation with 0.67 μM, 2.00 μM, 1.11 μM,and 6.00 μM concentrations of antisense oligonucleotide, as specified inTable 22. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and Target-X mRNA levels were measured byquantitative real-time PCR. Human Target-X primer probe set RTS2927 wasused to measure mRNA levels. Target-X mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of Target-X, relative to untreatedcontrol cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 22. As illustrated in Table 22, Target-X mRNAlevels were reduced in a dose-dependent manner in antisenseoligonucleotide treated cells. The data also confirms that several ofthe newly designed gapmers are more potent than ISIS 407939 of theprevious publication.

TABLE 22 Dose-dependent antisense inhibition of human Target-X in Hep3Bcells using electroporation 666.6667 2000.0 6000.0 IC₅₀ ISIS No nM nM nM(μM) 407939 47 68 85 0.7 457851 60 80 93 <0.6 472916 53 80 87 <0.6472925 62 86 95 <0.6 472926 66 77 85 <0.6 472935 54 84 94 <0.6 472958 6682 88 <0.6 472959 64 81 93 <0.6 472970 72 87 86 <0.6 472976 78 92 97<0.6 472994 79 92 96 <0.6 472995 61 82 93 <0.6 472996 73 91 95 <0.6472998 63 90 95 <0.6 473019 55 80 86 <0.6 473020 61 76 85 <0.6 473046 6180 94 <0.6 473055 55 84 94 <0.6 492104 53 76 88 <0.6 492105 62 80 90<0.6 492189 57 80 92 <0.6 492194 57 83 91 <0.6 492195 58 81 95 <0.6492196 62 86 95 <0.6 492205 62 87 95 <0.6 492215 60 78 89 <0.6 492221 6376 92 <0.6 492234 51 74 91 0.5 492276 50 56 95 0.8 492277 58 73 81 <0.6492306 61 75 84 <0.6 492317 59 80 93 <0.6 492329 59 70 89 <0.6 492331 6987 95 <0.6 492333 47 70 85 0.7 492334 57 77 90 <0.6 492390 72 88 95 <0.6492399 68 91 96 <0.6 492401 68 89 95 <0.6 492404 65 87 94 <0.6 492405 4481 90 0.7 492406 65 82 92 <0.6 492440 50 70 89 0.6 492465 16 80 79 1.4492467 58 77 92 <0.6 492492 45 80 94 0.7 492494 63 82 93 <0.6 492503 5581 93 <0.6 492530 70 86 90 <0.6 492534 67 85 91 <0.6 492536 54 81 89<0.6 492541 54 71 85 <0.6 492545 59 78 89 <0.6 492566 59 84 85 <0.6492571 52 81 89 <0.6 492572 67 83 90 <0.6 492573 69 83 92 <0.6 492574 6582 91 <0.6 492575 72 83 91 <0.6 492593 61 78 90 <0.6 492617 62 80 93<0.6 492618 47 79 94 0.6 492619 54 82 95 <0.6 492621 44 85 92 0.6 49265153 66 91 0.6 492652 61 78 88 <0.6 492658 59 79 88 <0.6 492725 43 84 890.6 492730 51 87 93 0.4 492731 46 82 90 0.6 492784 56 88 96 <0.6 49281668 89 97 <0.6 492818 64 84 96 <0.6 492877 67 91 93 <0.6 492878 80 89 93<0.6 492913 53 87 92 <0.6 492914 75 89 96 <0.6 492928 60 83 94 <0.6492938 70 90 92 <0.6 492991 67 93 99 <0.6 492992 0 82 95 2.1 493087 5481 90 <0.6 493114 50 73 90 0.6 493178 71 88 96 <0.6 493179 47 82 95 0.6493182 79 87 91 <0.6 493195 55 78 90 <0.6 493201 87 93 96 <0.6 493202 6889 94 <0.6 493255 57 79 93 <0.6 493291 57 87 93 <0.6 493292 70 89 93<0.6 493296 35 84 91 0.9 493298 57 84 92 <0.6 493299 65 84 93 <0.6493304 68 86 94 <0.6 493312 53 82 91 <0.6 493333 66 84 87 <0.6

Example 8 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3BCells

Additional antisense oligonucleotides from the studies described above,exhibiting in vitro inhibition of Target-X mRNA, were selected andtested at various doses in Hep3B cells. Cells were plated at a densityof 20,000 cells per well and transfected using electroporation with 0.67μM, 2.00 μM, 1.11 μM, and 6.00 μM concentrations of antisenseoligonucleotide, as specified in Table 23. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells and Target-XmRNA levels were measured by quantitative real-time PCR. Human Target-Xprimer probe set RTS2927 was used to measure mRNA levels. Target-X mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition of Target-X,relative to untreated control cells. As illustrated in Table 23,Target-X mRNA levels were reduced in a dose-dependent manner inantisense oligonucleotide treated cells. The data also confirms thatseveral of the newly designed gapmers are more potent than ISIS 407939.

TABLE 23 Dose-dependent antisense inhibition of human Target-X in Hep3Bcells using electroporation 0.67 2.00 6.00 IC₅₀ ISIS No μM μM μM (μM)407939 52 71 86 0.6 472983 49 83 97 0.5 472984 51 82 95 0.5 472991 49 8295 0.5 472998 59 88 96 <0.6 492365 74 91 96 <0.6 492377 56 76 91 <0.6492380 63 79 95 <0.6 492384 67 84 94 <0.6 492388 69 87 97 <0.6 492389 6290 96 <0.6 492391 56 84 94 <0.6 492398 63 80 95 <0.6 492403 58 81 91<0.6

Example 9 Modified Chimeric Antisense Oligonucleotides Comprising2′-Methoxyethyl (2′-MOE) Modifications at 5′ and 3′ Wing RegionsTargeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid and were tested for their effects on Target-X mRNA invitro. Also tested were ISIS 403052, ISIS 407939, ISIS 416446, ISIS416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130,and ISIS 422142 which were described in an earlier publication (WO2009/061851), incorporated herein by reference. ISIS 490149, ISIS490197, ISIS 490209, ISIS 490275, ISIS 490277, and ISIS 490424,described in the Examples above, were also included in the screen.

The newly designed chimeric antisense oligonucleotides in Table 24 weredesigned as 3-10-4 2′-MOE gapmers. These gapmers are 17 nucleosides inlength, wherein the central gap segment comprises of ten2′-deoxyribonucleosides and is flanked by wing segments on the 5′direction with three nucleosides and the 3′ direction with fournucleosides. Each nucleoside in the 5′ wing segment and each nucleosidein the 3′ wing segment has 2′-MOE modifications. The internucleosidelinkages throughout each gapmer are phosphorothioate (P═S) linkages. Allcytosine residues throughout each gapmer are 5-methylcytosines.

Each gapmer listed in Table 24 is targeted to the human Target-X genomicsequence.

Activity of the newly designed oligonucleotides was compared to ISIS403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508,ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142. Cultured Hep3Bcells at a density of 20,000 cells per well were transfected usingelectroporation with 2,000 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and Target-X mRNA levels were measured by quantitative real-timePCR. Human primer probe set RTS2927 (described hereinabove in Example 1)was used to measure mRNA levels. Target-X mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of Target-X, relative to untreatedcontrol cells. A total of 272 oligonucleotides were tested. Only thoseoligonucleotides which were selected for further studies are shown inTable 24. Several of the newly designed antisense oligonucleotidesprovided in Table 24 are more potent than antisense oligonucleotidesfrom the previous publication.

TABLE 24 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X ISIS No % inhibition Motif WingChemistry SEQ CODE 403052 51 5-10-5 2′-MOE 82 407939 78 5-10-5 2′-MOE 72416446 70 5-10-5 2′-MOE 103 416472 79 5-10-5 2′-MOE 111 416507 84 5-10-52′-MOE 97 416508 80 5-10-5 2′-MOE 100 422087 89 5-10-5 2′-MOE 121 42209678 5-10-5 2′-MOE 219 422130 81 5-10-5 2′-MOE 225 422142 84 5-10-5 2′-MOE99 490275 77 5-10-5 2′-MOE 90 513462 79 3-10-4 2′-MOE 213 513463 813-10-4 2′-MOE 214 490277 74 5-10-5 2′-MOE 91 513487 83 3-10-4 2′-MOE 215513504 81 3-10-4 2′-MOE 216 513507 86 3-10-4 2′-MOE 217 513508 85 3-10-42′-MOE 218 490424 69 5-10-5 2′-MOE 101 491122 87 5-10-5 2′-MOE 220513642 79 3-10-4 2′-MOE 221 490149 71 5-10-5 2′-MOE 109 513419 90 3-10-42′-MOE 222 513420 89 3-10-4 2′-MOE 223 513421 88 3-10-4 2′-MOE 224490197 77 5-10-5 2′-MOE 116 513446 89 3-10-4 2′-MOE 226 513447 83 3-10-42′-MOE 227 490209 79 5-10-5 2′-MOE 118 513454 84 3-10-4 2′-MOE 228513455 92 3-10-4 2′-MOE 229 513456 89 3-10-4 2′-MOE 230 513457 83 3-10-42′-MOE 231

Example 10 Dose-Dependent Antisense Inhibition of Human Target-X inHep3B Cells

Antisense oligonucleotides from the studies above, exhibiting in vitroinhibition of Target-X mRNA, were selected and tested at various dosesin Hep3B cells. ISIS 403052, ISIS 407643, ISIS 407935, ISIS 407936, ISIS407939, ISIS 416446, ISIS 416459, ISIS 416472, ISIS 416507, ISIS 416508,ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422130, ISIS and 422142,5-10-5 MOE gapmers targeting human Target-X, which were described in anearlier publication (WO 2009/061851).

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00μM concentrations of antisense oligonucleotide, as specified in Table25. After a treatment period of approximately 16 hours, RNA was isolatedfrom the cells and Target-X mRNA levels were measured by quantitativereal-time PCR. Human Target-X primer probe set RTS2927 was used tomeasure mRNA levels. Target-X mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of Target-X, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 25. As illustrated in Table 25, Target-X mRNAlevels were reduced in a dose-dependent manner in antisenseoligonucleotide treated cells. The data also confirms that the newlydesigned gapmers are potent than gapmers from the previous publication.

TABLE 25 Dose-dependent antisense inhibition of human Target-X in Hep3Bcells using electroporation 0.625 1.25 2.50 5.00 10.00 IC₅₀ ISIS No μMμM μM μM μM (μM) 403052 21 35 63 82 89 1.9 407643 29 46 67 83 90 1.4407935 52 68 80 89 91 <0.6 407936 31 51 62 78 84 1.4 407939 30 61 74 8388 1.0 416446 37 53 64 76 83 1.2 416459 51 76 83 90 92 <0.6 416472 37 5266 78 85 1.2 416507 45 68 82 87 90 0.7 416508 33 56 74 84 89 1.1 41654957 71 78 82 85 <0.6 422086 46 67 77 89 92 0.7 422087 50 69 74 86 91 0.6422130 32 65 78 92 93 0.9 422142 59 73 84 86 88 <0.6 490103 52 57 66 8388 0.9 490149 34 58 71 85 91 1.0 490196 26 59 66 79 84 1.3 490197 39 6374 81 90 0.8 490208 44 70 76 83 88 0.6 490275 36 58 76 85 89 1.0 49027737 63 73 87 87 0.8 490279 40 54 72 83 89 1.0 490323 49 68 79 86 90 <0.6490368 39 62 76 86 91 0.8 490396 36 53 69 80 87 1.1 490424 45 65 69 7682 0.6 490803 57 74 85 89 92 <0.6 513419 60 71 85 95 96 <0.6 513420 3769 79 94 96 0.7 513421 46 64 84 95 97 0.6 513446 47 81 88 95 96 <0.6513447 56 74 81 92 96 <0.6 513454 50 77 82 93 95 <0.6 513455 74 82 91 9696 <0.6 513456 66 80 88 94 95 <0.6 513457 54 67 80 87 89 <0.6 513462 4972 84 87 89 <0.6 513463 36 62 76 85 89 0.9 513487 42 56 73 87 93 0.9513504 47 65 81 90 91 0.6 513505 39 50 78 85 92 1.0 513507 52 73 83 8993 <0.6 513508 56 78 85 91 94 <0.6

Example 11 Dose-Dependent Antisense Inhibition of Human Target-X inHep3B Cells

Additional antisense oligonucleotides from the studies above, exhibitingin vitro inhibition of Target-X mRNA, were tested at various doses inHep3B cells. ISIS 407935, ISIS 407939, ISIS 416446, ISIS 416472, ISIS416507, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, and ISIS422142 5-10-5 MOE gapmers targeting human Target-X, which were describedin an earlier publication (WO 2009/061851).

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 0.3125 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00μM and 10.00 μM concentrations of antisense oligonucleotide, asspecified in Table 26. After a treatment period of approximately 16hours, RNA was isolated from the cells and Target-X mRNA levels weremeasured by quantitative real-time PCR. Human Target-X primer probe setRTS2927 was used to measure mRNA levels. Target-X mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of Target-X, relative tountreated control cells. As illustrated in Table 26, Target-X mRNAlevels were reduced in a dose-dependent manner in antisenseoligonucleotide treated cells. The data also confirms that the newlydesigned gapmers are more potent than gapmers from the previouspublication.

TABLE 26 Dose-dependent antisense inhibition of human Target-X in Hep3Bcells using electroporation 0.3125 0.625 1.250 2.500 5.000 10.000 IC₅₀ISIS No μM μM μM μM μM μM (μM) 407935 30 49 75 86 91 94 0.6 407939 30 4861 78 85 90 0.8 416446 27 52 63 75 85 90 0.7 416472 38 51 72 83 88 940.5 416507 58 81 76 84 89 92 <0.3 416549 52 67 75 81 88 89 0.3 422086 4849 68 78 86 91 0.5 422087 30 56 66 83 72 92 0.6 422096 47 63 70 77 83 85<0.3 422142 69 85 87 85 89 91 <0.3 490103 52 57 68 78 87 93 0.4 49014933 64 62 77 86 93 0.5 490197 38 46 60 75 87 93 0.7 490208 46 62 73 83 8891 0.4 490209 40 54 72 79 85 94 0.5 490275 52 61 67 78 85 91 0.3 49027733 59 77 79 91 94 0.5 490323 43 61 72 69 84 87 0.4 490368 50 64 78 83 9092 <0.3 490396 46 64 68 84 84 90 0.3 490424 24 47 58 72 76 82 1.0 49080345 60 70 84 88 89 0.3 513419 32 53 76 88 93 95 0.5 513420 35 59 72 82 9497 0.5 513421 46 67 78 86 94 96 <0.3 513446 26 61 77 89 91 97 0.5 51344722 48 60 82 91 95 0.8 513454 25 59 76 86 94 96 0.5 513455 60 73 85 89 9596 <0.3 513456 49 60 81 88 94 95 <0.3 513457 43 50 72 77 87 92 0.5513462 25 48 58 76 83 88 0.8 513463 22 45 66 73 85 88 0.9 513487 41 5665 79 86 90 0.4 513504 19 48 63 76 87 92 0.9 513505 11 21 54 73 85 901.4 513507 47 55 72 82 90 91 0.3 513508 31 59 74 85 92 93 0.5 513642 4355 67 80 88 92 0.4

Example 12 Tolerability of 2′-MOE Gapmers Targeting Human Target-X inBALB/c Mice

BALB/c mice are a multipurpose mice model, frequently utilized forsafety and efficacy testing. The mice were treated with ISIS antisenseoligonucleotides selected from studies described above and evaluated forchanges in the levels of various plasma chemistry markers.

Treatment

Groups of male BALB/c mice were injected subcutaneously twice a week for3 weeks with 50 mg/kg of ISIS 407935, ISIS 416472, ISIS 416549, ISIS422086, ISIS 422087, ISIS 422096, ISIS 422142, ISIS 490103, ISIS 490149,ISIS 490196, ISIS 490208, ISIS 490209, ISIS 513419, ISIS 513420, ISIS513421, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513462,ISIS 513463, ISIS 513487, ISIS 513504, ISIS 513508, and ISIS 513642. Onegroup of male BALB/c mice was injected subcutaneously twice a week for 3weeks with PBS. Mice were euthanized 48 hours after the last dose, andorgans and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels oftransaminases, or which caused an increase within three times the upperlimit of normal (ULN) were deemed very tolerable. ISIS oligonucleotidesthat caused an increase in the levels of transaminases between threetimes and seven times the ULN were deemed tolerable. Based on thesecriteria, ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS422096, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513454, ISIS 513455,ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513504, and ISIS 513508 wereconsidered very tolerable in terms of liver function. Based on thesecriteria, ISIS 422086, ISIS 490209, ISIS 513419, ISIS 513420, and ISIS513463 were considered tolerable in terms of liver function.

Example 13 Dose-Dependent Antisense Inhibition of Human Target-X inHep3B cells

Additional antisense oligonucleotides from the studies above, exhibitingin vitro inhibition of Target-X mRNA were selected and tested at variousdoses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer,which was described in an earlier publication (WO 2009/061851).

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 0.074 μM, 0.222 μM, 0.667 μM, 2.000 μM, and6.000 μM concentrations of antisense oligonucleotide, as specified inTable 27. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and Target-X mRNA levels were measured byquantitative real-time PCR. Human Target-X primer probe set RTS2927(described hereinabove in Example 1) was used to measure mRNA levels.Target-X mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN®. Results are presented as percent inhibition ofTarget-X, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 27. As illustrated in Table 27, Target-X mRNAlevels were reduced in a dose-dependent manner in antisenseoligonucleotide treated cells. Many of the newly designed antisenseoligonucleotides provided in Table 27 achieved an IC₅₀ of less than 0.9μM and, therefore, are more potent than ISIS 407939.

TABLE 27 Dose-dependent antisense inhibition of human Target-X in Hep3Bcells using electroporation 0.074 0.222 0.667 2.000 6.000 IC₅₀ ISIS NoμM μM μM μM μM (μM) 407939 2 17 53 70 87 0.9 472970 17 47 75 92 95 0.3472988 0 8 21 54 92 1.4 472996 18 59 74 93 95 0.2 473244 91 95 97 99 99<0.07 473286 6 53 85 92 98 0.3 473359 2 3 20 47 67 2.6 473392 71 85 8892 96 <0.07 473393 91 96 97 98 99 <0.07 473547 85 88 93 97 98 <0.07473567 0 25 66 88 95 0.7 473589 8 47 79 94 99 0.3 482814 23 68 86 93 960.1 482815 6 48 65 90 96 0.4 482963 3 68 85 94 96 0.2 483241 14 33 44 7693 0.6 483261 14 21 41 72 88 0.7 483290 0 1 41 69 92 1.0 483414 8 1 3676 91 0.9 483415 0 40 52 84 94 0.6 484559 26 51 78 87 97 0.2 484713 6 553 64 88 0.9

Example 14 Modified Antisense Oligonucleotides Comprising2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt)Modifications Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid and were tested for their effects on Target-X mRNA invitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting humanTarget-X, which was described in an earlier publication (WO2009/061851). ISIS 472998, ISIS 492878, and ISIS 493201 and 493182,2-10-2 cEt gapmers, described in the Examples above were also includedin the screen.

The newly designed modified antisense oligonucleotides are 16nucleotides in length and their motifs are described in Table 28. Thechemistry column of Table 28 presents the sugar motif of eacholigonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE)nucleoside, “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g cEt) and“d” indicates a 2′-deoxyribonucleoside. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytosineresidues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 28 is targeted to the human Target-X genomicsequence.

Activity of newly designed gapmers was compared to ISIS 407939. CulturedHep3B cells at a density of 20,000 cells per well were transfected usingelectroporation with 2,000 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and Target-X mRNA levels were measured by quantitative real-timePCR. Human primer probe set RTS2927 was used to measure mRNA levels.Target-X mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN®. Results are presented as percent inhibition ofTarget-X, relative to untreated control cells and demonstrate thatseveral of the newly designed gapmers are more potent than ISIS 407939.A total of 685 oligonucleotides were tested. Only those oligonucleotideswhich were selected for further studies are shown in Table 28.

TABLE 28 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X ISIS No % inhibition Chemistry SEQCODE 407939 68 eeeee-d(10)-eeeee 72 492878 73 kk-d(10)-kk 493182 80kk-d(10)-kk 493201 84 kk-d(10)-kk 472998 91 kk-d(10)-kk 515640 75eee-d(10)-kkk 23 515637 77 eee-d(10)-kkk 232 515554 72 eee-d(10)-kkk 233515406 80 kkk-d(10)-eee 234 515558 81 eee-d(10)-kkk 234 515407 88kkk-d(10)-eee 235 515408 85 kkk-d(10)-eee 236 515422 86 kkk-d(10)-eee237 515423 90 kkk-d(10)-eee 238 515575 84 eee-d(10)-kkk 238 515424 87kkk-d(10)-eee 239 515432 78 kkk-d(10)-eee 240 515433 71 kkk-d(10)-eee241 515434 76 kkk-d(10)-eee 242 515334 85 kkk-d(10)-eee 243 515649 61eee-d(10)-kkk 88 515338 86 kkk-d(10)-eee 244 515438 76 kkk-d(10)-eee 245515439 75 kkk-d(10)-eee 246 516003 87 eee-d(10)-kkk 247 515647 60eee-d(10)-kkk 77 515639 78 eee-d(10)-kkk 34 493201 84 eee-d(10)-kkk 202515648 36 kkk-d(10)-eee 248 515641 69 kk-d(10)-eeee 39 515650 76kkk-d(10)-eee 44 515354 87 eee-d(10)-kkk 249 515926 87 eee-d(10)-kkk 250515366 87 kk-d(10)-eeee 251 515642 58 kkk-d(10)-eee 252 515643 81eee-d(10)-kkk 53 515944 84 kk-d(10)-eeee 253 515380 90 kkk-d(10)-eee 254515532 83 kkk-d(10)-eee 254 515945 85 kk-d(10)-eeee 254 515381 82kk-d(10)-eeee 255 515382 95 kkk-d(10)-eee 256 515948 94 eee-d(10)-kkk256 515949 87 eee-d(10)-kkk 257 515384 89 kkk-d(10)-eee 258 515635 82kk-d(10)-eeee 65 515638 90 kkk-d(10)-eee 67 515386 92 kk-d(10)-eeee 259515951 84 eee-d(10)-kkk 259 515387 78 kkk-d(10)-eee 260 515952 89kkk-d(10)-eee 260 515636 90 kkk-d(10)-eee 69 515388 84 eee-d(10)-kkk 261e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 15 Tolerability of Modified Oligonucleotides Comprising2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt)Modifications Targeting Human Target-X in BALB/c Mice

BALB/c mice were treated with ISIS antisense oligonucleotides selectedfrom studies described above and evaluated for changes in the levels ofvarious plasma chemistry markers.

Additionally, the newly designed modified antisense oligonucleotideswere also added to this screen. The newly designed chimeric antisenseoligonucleotides are 16 nucleotides in length and their motifs aredescribed in Table 29. The chemistry column of Table 29 presents thesugar motif of each oligonucleotide, wherein “e” indicates a2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH₃bicyclic nucleoside (e.g cEt) and “d” indicates a2′-deoxyribonucleoside. The internucleoside linkages throughout eachgapmer are phosphorothioate (P═S) linkages. All cytosine residuesthroughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 29 is targeted to the human Target-X genomicsequence.

TABLE 29 Modified chimeric antisense oligonucleotides targeted toTarget-X ISIS No Chemistry SEQ CODE 516044 eee-d(10)-kkk 21 516045eee-d(10)-kkk 22 516058 eee-d(10)-kkk 26 516059 eee-d(10)-kkk 27 516060eee-d(10)-kkk 28 516061 eee-d(10)-kkk 29 516062 eee-d(10)-kkk 30 516046eee-d(10)-kkk 37 516063 eee-d(10)-kkk 38 516064 eee-d(10)-kkk 89 516065eee-d(10)-kkk 262 516066 eee-d(10)-kkk 263 516047 eee-d(10)-kkk 41516048 eee-d(10)-kkk 42 516049 eee-d(10)-kkk 81 516050 eee-d(10)-kkk 45516051 eee-d(10)-kkk 48 516052 eee-d(10)-kkk 49 515652 eee-d(10)-kkk 50508039 eee-d(10)-kkk 264 516053 eee-d(10)-kkk 265 515654 eee-d(10)-kkk76 515656 eee-d(10)-kkk 77 516054 eee-d(10)-kkk 57 516055 eee-d(10)-kkk59 515655 eee-d(10)-kkk 61 516056 eee-d(10)-kkk 63 516057 eee-d(10)-kkk64 515653 eee-d(10)-kkk 71 515657 eee-d(10)-kkk 73 e = 2′-MOE, k = cEt,d = 2′-deoxyribonucleoside

Treatment

Groups of 4-6-week old male BALB/c mice were injected subcutaneouslytwice a week for 3 weeks with 50 mg/kg/week of ISIS 457851, ISIS 515635,ISIS 515636, ISIS 515637, ISIS 515638, ISIS 515639, ISIS 515640, ISIS515641, ISIS 515642, ISIS 515643, ISIS 515647, ISIS 515648, ISIS 515649,ISSI 515650, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS515656, ISIS 515657, ISIS 516044, ISIS 516045, ISIS 516046, ISIS 516047,ISIS 516048, ISIS 516049, ISIS 516050, ISIS 516051, ISIS 516052, ISIS516053, ISIS 516054, ISIS 516055, ISIS 516056, ISIS 516057, ISIS 516058,ISIS 516059, ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS516064, ISIS 516065, and ISIS 516066. One group of 4-6-week old maleBALB/c mice was injected subcutaneously twice a week for 3 weeks withPBS. Mice were euthanized 48 hours after the last dose, and organs andplasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels oftransaminases, or which caused an increase within three times the upperlimit of normal (ULN) were deemed very tolerable. ISIS oligonucleotidesthat caused an increase in the levels of transaminases between threetimes and seven times the ULN were deemed tolerable. Based on thesecriteria, ISIS 515636, ISIS 515639, ISIS 515641, ISIS 515642, ISIS515648, ISIS 515650, ISIS 515652, ISIS 515653, ISIS 515655, ISIS 515657,ISIS 516044, ISIS 516045, ISIS 516047, ISIS 516048, ISIS 516051, ISIS516052, ISIS 516053, ISIS 516055, ISIS 516056, ISIS 516058, ISIS 516059,ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS 516064, ISIS516065, and ISIS 516066 were considered very tolerable in terms of liverfunction. Based on these criteria, ISIS 457851, ISIS 515635, ISIS515637, ISIS 515638, ISIS 515643, ISIS 515647, ISIS 515649, ISIS 515650,ISIS 515652, ISIS 515654, ISIS 515656, ISIS 516056, and ISIS 516057 wereconsidered tolerable in terms of liver function.

Example 16 Efficacy of Modified Oligonucleotides Comprising2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt)Modifications Targeting Human Target-X in Transgenic Mice

Transgenic mice were developed at Taconic farms harboring a Target-Xgenomic DNA fragment. The mice were treated with ISIS antisenseoligonucleotides selected from studies described above and evaluated forefficacy.

Treatment

Groups of 3-4 male and female transgenic mice were injectedsubcutaneously twice a week for 3 weeks with 20 mg/kg/week of ISIS457851, ISIS 515636, ISIS 515639, ISIS 515653, ISIS 516053, ISIS 516065,and ISIS 516066. One group of mice was injected subcutaneously twice aweek for 3 weeks with control oligonucleotide, ISIS 141923(CCTTCCCTGAAGGTTCCTCC, 5-10-5 MOE gapmer with no known murine target,SEQ ID NO: 9). One group of mice was injected subcutaneously twice aweek for 3 weeks with PBS. Mice were euthanized 48 hours after the lastdose, and organs and plasma were harvested for further analysis.

RNA Analysis

RNA was extracted from plasma for real-time PCR analysis of Target-X,using primer probe set RTS2927. The mRNA levels were normalized usingRIBOGREEN®. Results are presented as percent inhibition of Target-X,relative to control. As shown in Table 30, each of the antisenseoligonucleotides achieved reduction of human Target-X mRNA expressionover the PBS control. Treatment with the control oligonucleotide did notachieve reduction in Target-X levels, as expected.

TABLE 30 Percent inhibition of Target-X mRNA in transgenic mice ISIS No% inhibition 141923 0 457851 76 515636 66 515639 49 515653 78 516053 72516065 59 516066 39

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISAkit (purchased from Hyphen Bio-Med). Results are presented as percentinhibition of Target-X, relative to control. As shown in Table 31,several antisense oligonucleotides achieved reduction of human Target-Xprotein expression over the PBS control.

TABLE 31 Percent inhibition of Target-X protein levels in transgenicmice ISIS No % inhibition 141923 0 457851 64 515636 68 515639 46 5156530 516053 19 516065 0 516066 7

Example 17 Efficacy of Modified Oligonucleotides Comprising2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt)Modifications Targeting Human Target-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotidesselected from studies described above and evaluated for efficacy.

Treatment

Groups of 2-4 male and female transgenic mice were injectedsubcutaneously twice a week for 3 weeks with 10 mg/kg/week of ISIS407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 473137,ISIS 473244, ISIS 473326, ISIS 473327, ISIS 473359, ISIS 473392, ISIS473393, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559,ISIS 484713, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513419, ISIS513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513508,ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515655, ISIS515657, ISIS 516045, ISIS 516046, ISIS 516047, ISIS 516048, ISIS 516051,ISIS 516052, ISIS 516055, ISIS 516056, ISIS 516059, ISIS 516061, ISIS516062, and ISIS 516063. One group of mice was injected subcutaneouslytwice a week for 3 weeks with PBS. Mice were euthanized 48 hours afterthe last dose, and organs and plasma were harvested for furtheranalysis.

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISAkit (purchased from Hyphen Bio-Med). Results are presented as percentinhibition of Target-X, relative to control. As shown in Table 32,several antisense oligonucleotides achieved reduction of human Target-Xover the PBS control.

TABLE 32 Percent inhibition of Target-X plasma protein levels intransgenic mice ISIS No % inhibition 407935 80 416472 49 416549 29422087 12 422096 21 473137 57 473244 67 473326 42 473327 100 473359 0473392 22 473393 32 473547 73 473567 77 473589 96 473630 75 484559 75484713 56 490103 0 490196 74 490208 90 513419 90 513454 83 513455 91513456 81 513457 12 513487 74 513508 77 515640 83 515641 87 515642 23515648 32 515655 79 515657 81 516045 52 516046 79 516047 65 516048 79516051 84 516052 72 516055 70 516056 0 516059 39 516061 64 516062 96516063 24

Example 18 Dose-Dependent Antisense Inhibition of Human Target-X inHep3B Cells

Antisense oligonucleotides exhibiting in vitro inhibition of Target-XmRNA were selected and tested at various doses in Hep3B cells. Alsotested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X,which was described in an earlier publication (WO 2009/061851).

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 0.074 μM, 0.222 μM, 0.667 μM, 2.000 μM, and6.000 μM concentrations of antisense oligonucleotide, as specified inTable 33. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and Target-X mRNA levels were measured byquantitative real-time PCR. Human Target-X primer probe set RTS2927(described hereinabove in Example 1) was used to measure mRNA levels.Target-X mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN®. Results are presented as percent inhibition ofTarget-X, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 33. As illustrated in Table 33, Target-X mRNAlevels were reduced in a dose-dependent manner in antisenseoligonucleotide treated cells. Many of the newly designed antisenseoligonucleotides provided in Table 33 achieved an IC₅₀ of less than 2.0μM and, therefore, are more potent than ISIS 407939.

TABLE 33 Dose-dependent antisense inhibition of human Target-X in Hep3Bcells using electroporation 0.074 0.222 0.667 2.000 6.000 IC₅₀ ISIS NoμM μM μM μM μM (μM) 407939 0 9 21 58 76 2.0 515636 14 32 50 62 81 0.7515639 10 24 41 61 67 1.3 515640 4 16 35 52 63 2.0 515641 0 21 27 55 661.9 515642 3 13 36 44 66 2.2 515648 8 10 10 5 16 >6.0 515653 9 35 26 5571 1.5 515655 0 0 6 13 42 >6.0 515657 0 13 17 38 51 6.0 516045 0 6 15 1940 >6.0 516046 0 7 32 48 69 2.1 516047 12 27 41 50 63 1.8 516051 9 8 3452 66 2.0 516052 17 42 27 53 75 1.2 516053 9 7 28 63 77 1.3 516055 0 327 54 75 2.0 516056 0 4 14 52 66 2.6 516057 0 34 33 51 70 1.6 516058 1312 25 47 74 2.0 516059 4 15 36 47 68 1.9 516060 0 1 39 29 63 3.2 5160610 0 24 0 3 <6.0 516062 0 20 43 65 78 1.0 516063 0 8 10 37 61 3.8 5160640 3 13 45 69 2.7 516065 0 14 38 63 76 1.3 516066 0 3 30 55 75 1.7

Example 19 Modified Oligonucleotides Comprising 2′-O-Methoxyethyl(2′-MOE) and 6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt) ModificationsTargeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid and were tested for their effects on Target-X mRNA invitro. ISIS 472998, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655,ISIS 515656, and ISIS 515657, described in the Examples above were alsoincluded in the screen.

The newly designed chimeric antisense oligonucleotides are 16 or 17nucleotides in length and their motifs are described in Table 34. Thechemistry column of Table 34 presents the sugar motif of eacholigonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE)nucleoside, “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g cEt) and“d” indicates a 2′-deoxyribonucleoside. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytosineresidues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 34 is targeted to the human Target-X genomicsequence.

Activity of newly designed gapmers was compared to ISIS 407939. CulturedHep3B cells at a density of 20,000 cells per well were transfected usingelectroporation with 2,000 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and Target-X mRNA levels were measured by quantitative real-timePCR. Human primer probe set RTS2927 (described hereinabove in Example 1)was used to measure mRNA levels. Target-X mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of Target-X, relative to untreatedcontrol cells.

TABLE 34 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X ISIS No % inhibition Chemistry SEQCODE 472998 85 kk-d(10)-kk 74 515652 63 eee-d(10)-kkk 50 515653 67eee-d(10)-kkk 71 515654 78 eee-d(10)-kkk 86 515655 41 eee-d(10)-kkk 61515656 74 eee-d(10)-kkk 87 515657 49 eee-d(10)-kkk 73 529265 52eek-d(10)-keke 267 529332 82 eek-d(10)-keke 268 529334 78 eek-d(10)-keke269 529186 85 eek-d(10)-keke 213 529223 81 eek-d(10)-kkke 213 529129 75eee-d(10)-kkk 270 529149 82 kkk-d(10)-eee 270 529177 77 eek-d(10)-keke214 529214 78 eek-d(10)-kkke 214 529178 79 eek-d(10)-keke 271 529215 82eek-d(10)-kkke 271 529179 71 eek-d(10)-keke 272 529216 77 eek-d(10)-kkke272 529193 69 eek-d(10)-keke 273 529230 70 eek-d(10)-kkke 273 529136 48eee-d(10)-kkk 274 529156 68 kkk-d(10)-eee 274 529194 44 eek-d(10)-keke275 529231 56 eek-d(10)-kkke 275 529137 34 eee-d(10)-kkk 276 529157 79kkk-d(10)-eee 276 529336 57 eek-d(10)-keke 277 529338 73 eek-d(10)-keke278 529195 55 eek-d(10)-keke 279 529232 68 eek-d(10)-kkke 279 529340 65eek-d(10)-keke 280 529342 69 eek-d(10)-keke 281 529812 69 k-d(10)-kekee282 529831 62 k-d(10)-kdkee 282 529733 64 ke-d(10)-keke 283 529753 52ek-d(10)-keke 283 529773 57 ke-d(10)-kdke 283 529793 36 ek-d(10)-kdke283 529862 48 kde-d(10)-kdke 284 529882 35 edk-d(10)-kdke 284 529902 44k-(d4)-k-(d4)-k-(d4)-ke 284 529559 71 eek-d(10)-kke 26 529584 57kee-d(10)-kke 26 529609 58 edk-d(10)-kke 26 529634 49 kde-d(10)-kke 26529659 52 kddk-d(9)-kke 26 529684 48 kdde-d(9)-kke 26 529709 61eddk-d(9)-kke 26 529922 52 eeee-d(9)-kke 26 529344 50 eek-d(10)-keke 285529138 32 eee-d(10)-kkk 286 529158 75 kkk-d(10)-eee 286 529184 75eek-d(10)-keke 215 529221 78 eek-d(10)-kkke 215 529127 67 eee-d(10)-kkk287 529147 79 kkk-d(10)-eee 287 529346 58 eek-d(10)-keke 288 529348 65eek-d(10)-keke 289 529350 77 eek-d(10)-keke 290 529813 20 k-d(10)-kekee291 529832 47 k-d(10)-kdkee 291 529734 63 ke-d(10)-keke 292 529754 58ek-d(10)-keke 292 529774 49 ke-d(10)-kdke 292 529794 51 ek-d(10)-kdke292 529863 64 kde-d(10)-kdke 293 529883 78 edk-d(10)-kdke 293 529903 36k-d(4)-k-d(4)-k-d(4)-ke 293 529560 71 eek-d(10)-kke 27 529585 70kee-d(10)-kke 27 529610 66 edk-d(10)-kke 27 529635 45 kde-d(10)-kke 27529660 53 kddk-d(9)-kke 27 529685 42 kdde-d(9)-kke 27 529710 60eddk-d(9)-kke 27 529923 63 eeee-d(9)-kke 27 529196 74 eek-d(10)-keke 294529233 80 eek-d(10)-kkke 294 529139 75 eee-d(10)-kkk 295 529159 62kkk-d(10)-eee 295 529352 74 eek-d(10)-keke 296 529354 67 eek-d(10)-keke297 529197 43 eek-d(10)-keke 298 529234 58 eek-d(10)-kkke 298 529140 29eee-d(10)-kkk 299 529160 59 kkk-d(10)-eee 299 529180 80 eek-d(10)-keke216 529217 79 eek-d(10)-kkke 216 529814 51 k-d(10)-kekee 300 529833 52k-d(10)-kdkee 300 529735 43 ke-d(10)-keke 301 529755 60 ek-d(10)-keke301 529775 38 ke-d(10)-kdke 301 529795 58 ek-d(10)-kdke 301 529864 41kde-d(10)-kdke 302 529884 48 edk-d(10)-kdke 302 529904 44k-d(4)-k-(d4)-k-d(4)-ke 302 529934 61 eek-d(10)-keke 302 529356 71eek-d(10)-keke 303 529561 75 eek-d(10)-kke 28 529586 65 kee-d(10)-kke 28529611 54 edk-d(10)-kke 28 529636 39 kde-d(10)-kke 28 529661 67kddk-d(9)-kke 28 529686 66 kdde-d(9)-kke 28 529711 60 eddk-d(9)-kke 28529924 62 eeee-d(9)-kke 28 529358 82 eek-d(10)-keke 304 529181 79eek-d(10)-keke 217 529218 73 eek-d(10)-kkke 217 529182 85 eek-d(10)-keke218 529219 84 eek-d(10)-kkke 218 529360 84 eek-d(10)-keke 305 529362 87eek-d(10)-keke 306 529364 81 eek-d(10)-keke 307 529366 77 eek-d(10)-keke308 529198 28 eek-d(10)-keke 309 529235 8 eek-d(10)-kkke 309 529141 34eee-d(10)-kkk 310 529161 66 kkk-d(10)-eee 310 529368 27 eek-d(10)-keke311 529370 44 eek-d(10)-keke 312 529372 61 eek-d(10)-keke 313 529374 71eek-d(10)-keke 314 529376 63 eek-d(10)-keke 315 529378 68 eek-d(10)-keke316 529380 79 eek-d(10)-keke 317 529382 77 eek-d(10)-keke 318 529384 75eek-d(10)-keke 319 529386 40 eek-d(10)-keke 320 529240 73 eek-d(10)-keke321 529241 67 eek-d(10)-keke 322 529242 42 eek-d(10)-keke 323 529243 60eek-d(10)-keke 324 529388 65 eek-d(10)-keke 325 529815 37 k-d(10)-kekee326 529834 44 k-d(10)-kdkee 326 529736 47 ke-d(10)-keke 327 529756 78ek-d(10)-keke 327 529776 37 ke-d(10)-kdke 327 529796 71 ek-d(10)-kdke327 529865 70 kde-d(10)-kdke 328 529885 59 edk-d(10)-kdke 328 529905 54k-(d4)-k-(d4)-k-(d4)-ke 328 529935 70 eek-d(10)-keke 328 529562 87eek-d(10)-kke 29 529587 68 kee-d(10)-kke 29 529612 67 edk-d(10)-kke 29529637 64 kde-d(10)-kke 29 529662 62 kddk-d(9)-kke 29 529687 63kdde-d(9)-kke 29 529712 61 eddk-d(9)-kke 29 529925 61 eeee-d(9)-kke 29529816 77 k-d(10)-kekee 329 529835 80 k-d(10)-kdkee 329 529737 82ke-d(10)-keke 330 529757 83 ek-d(10)-keke 330 529777 68 ke-d(10)-kdke330 529797 77 ek-d(10)-kdke 330 529866 15 kde-d(10)-kdke 331 529886 71edk-d(10)-kdke 331 529906 63 k-(d4)-k-(d4)-k-(d4)-ke 331 529936 78eek-d(10)-keke 331 529563 89 eek-d(10)-kke 30 529588 84 kee-d(10)-kke 30529613 80 edk-d(10)-kke 30 529638 48 kde-d(10)-kke 30 529663 85kddk-d(9)-kke 30 529688 42 kdde-d(9)-kke 30 529713 81 eddk-d(9)-kke 30529926 67 eeee-d(9)-kke 30 529390 53 eek-d(10)-keke 332 529392 63eek-d(10)-keke 333 529394 58 eek-d(10)-keke 334 529396 56 eek-d(10)-keke335 529398 62 eek-d(10)-keke 336 529400 44 eek-d(10)-keke 337 529402 39eek-d(10)-keke 338 529404 46 eek-d(10)-keke 339 529406 63 eek-d(10)-keke340 529244 58 eek-d(10)-keke 341 529245 68 eek-d(10)-keke 342 529246 60eek-d(10)-keke 343 529247 36 eek-d(10)-keke 344 529248 43 eek-d(10)-keke345 529249 23 eek-d(10)-keke 346 529250 69 eek-d(10)-keke 347 529251 15eek-d(10)-keke 348 529252 44 eek-d(10)-keke 349 529253 42 eek-d(10)-keke350 529408 67 eek-d(10)-keke 351 529410 19 eek-d(10)-keke 352 529412 57eek-d(10)-keke 353 529414 80 eek-d(10)-keke 354 529416 85 eek-d(10)-keke355 529418 70 eek-d(10)-keke 356 529420 78 eek-d(10)-keke 357 529422 19eek-d(10)-keke 358 529424 48 eek-d(10)-keke 359 529426 66 eek-d(10)-keke360 529428 59 eek-d(10)-keke 361 529430 83 eek-d(10)-keke 362 529432 84eek-d(10)-keke 363 529199 71 eek-d(10)-keke 364 529236 76 eek-d(10)-kkke364 529142 64 eee-d(10)-kkk 365 529162 60 kkk-d(10)-eee 365 529254 46eek-d(10)-keke 366 529255 52 eek-d(10)-keke 367 529256 57 eek-d(10)-keke368 529257 55 eek-d(10)-keke 369 529258 3 eek-d(10)-keke 370 529259 71eek-d(10)-keke 371 529260 72 eek-d(10)-keke 372 529261 56 eek-d(10)-keke373 529262 56 eek-d(10)-keke 374 529263 59 eek-d(10)-keke 375 529264 49eek-d(10)-keke 376 529434 83 eek-d(10)-keke 377 529436 80 eek-d(10)-keke378 529438 79 eek-d(10)-keke 379 529440 87 eek-d(10)-keke 380 529442 68eek-d(10)-keke 381 529443 72 eek-d(10)-keke 382 529444 68 eek-d(10)-keke383 529445 85 eek-d(10)-keke 384 529446 72 eek-d(10)-keke 385 529447 60eek-d(10)-keke 386 529448 77 eek-d(10)-keke 387 529807 78 k-d(10)-kekee388 529826 61 k-d(10)-kdkee 388 529449 81 eek-d(10)-keke 389 529728 75ke-d(10)-keke 390 529748 80 ek-d(10)-keke 390 529768 68 ke-d(10)-kdke390 529788 74 ek-d(10)-kdke 390 529857 67 kde-d(10)-kdke 389 529877 77edk-d(10)-kdke 389 529897 26 k-(d4)-k-(d4)-k-(d4)-ke 389 529200 78eek-d(10)-keke 391 529237 84 eek-d(10)-kkke 391 529564 90 eek-d(10)-kke34 529589 86 kee-d(10)-kke 34 529614 82 edk-d(10)-kke 34 529639 80kde-d(10)-kke 34 529664 69 kddk-d(9)-kke 34 529689 71 kdde-d(9)-kke 34529714 73 eddk-d(9)-kke 34 529917 73 eeee-d(9)-kke 34 529143 68eee-d(10)-kkk 392 529163 50 kkk-d(10)-eee 392 529201 76 eek-d(10)-keke393 529238 72 eek-d(10)-kkke 393 529144 57 eee-d(10)-kkk 394 529164 71kkk-d(10)-eee 394 529450 91 eek-d(10)-keke 395 529451 85 eek-d(10)-keke396 529266 63 eek-d(10)-keke 397 529806 52 k-d(10)-kekee 398 529825 44k-d(10)-kdkee 398 529267 56 eek-d(10)-keke 399 529727 67 ke-d(10)-keke400 529747 63 ek-d(10)-keke 400 529767 67 ke-d(10)-kdke 400 529787 68ek-d(10)-kdke 400 529856 42 kde-d(10)-kdke 399 529876 36 edk-d(10)-kdke399 529896 56 k-(d4)-k-(d4)-k-(d4)-ke 399 529546 65 eek-d(10)-kke 248529571 80 kee-d(10)-kke 248 529596 43 edk-d(10)-kke 248 529621 38kde-d(10)-kke 248 529646 68 kddk-d(9)-kke 248 529671 50 kdde-d(9)-kke248 529696 53 eddk-d(9)-kke 248 529916 22 eeee-d(9)-kke 248 529547 86eek-d(10)-kke 37 529572 75 kee-d(10)-kke 37 529597 58 edk-d(10)-kke 37529622 58 kde-d(10)-kke 37 529647 18 kddk-d(9)-kke 37 529672 23kdde-d(9)-kke 37 529697 28 eddk-d(9)-kke 37 529928 36 eeee-d(9)-kke 37529452 63 eek-d(10)-keke 401 529453 73 eek-d(10)-keke 402 529454 82eek-d(10)-keke 403 529455 84 eek-d(10)-keke 404 529202 61 eek-d(10)-keke405 529239 59 eek-d(10)-kkke 405 529145 54 eee-d(10)-kkk 406 529165 77kkk-d(10)-eee 406 529456 69 eek-d(10)-keke 407 529457 81 eek-d(10)-keke408 529458 72 eek-d(10)-keke 409 529459 86 eek-d(10)-keke 410 529460 88eek-d(10)-keke 411 529817 46 k-d(10)-kekee 412 529836 49 k-d(10)-kdkee412 529738 51 ke-d(10)-keke 413 529758 53 ek-d(10)-keke 413 529778 39ke-d(10)-kdke 413 529798 52 ek-d(10)-kdke 413 529867 56 kde-d(10)-kdke414 529887 68 edk-d(10)-kdke 414 529907 28 k-(d4)-k-(d4)-k-(d4)-ke 414529938 64 eek-d(10)-keke 414 529565 81 eek-d(10)-kke 38 529590 49kee-d(10)-kke 38 529615 65 edk-d(10)-kke 38 529640 54 kde-d(10)-kke 38529665 77 kddk-d(9)-kke 38 529690 77 kdde-d(9)-kke 38 529715 63eddk-d(9)-kke 38 529927 62 eeee-d(9)-kke 38 529185 66 eek-d(10)-keke 221529222 62 eek-d(10)-kkke 221 529808 75 k-d(10)-kekee 89 529827 67k-d(10)-kdkee 89 529128 64 eee-d(10)-kkk 415 529148 78 kkk-d(10)-eee 415529461 87 eek-d(10)-keke 416 529729 71 ke-d(10)-keke 415 529749 83ek-d(10)-keke 415 529769 63 ke-d(10)-kdke 415 529789 10 ek-d(10)-kdke415 529800 69 k-d(10)-kekee 415 529819 78 k-d(10)-kdkee 415 529858 60kde-d(10)-kdke 416 529878 75 edk-d(10)-kdke 416 529898 34k-(d4)-k-(d4)-k-(d4)-ke 416 529566 61 eek-d(10)-kke 39 529591 71kee-d(10)-kke 39 529616 71 edk-d(10)-kke 39 529641 65 kde-d(10)-kke 39529666 70 kddk-d(9)-kke 39 529691 67 kdde-d(9)-kke 39 529716 75eddk-d(9)-kke 39 529721 71 ke-d(10)-keke 39 529741 81 ek-d(10)-keke 39529761 66 ke-d(10)-kdke 39 529781 65 ek-d(10)-kdke 39 529801 71k-d(10)-kekee 39 529820 74 k-d(10)-kdkee 39 529850 63 kde-d(10)-kdke 417529870 72 edk-d(10)-kdke 417 529890 23 k-(d4)-k-(d4)-k-(d4)-ke 417529918 54 eeee-d(9)-kke 39 529567 75 eek-d(10)-kke 262 529592 80kee-d(10)-kke 262 529617 65 edk-d(10)-kke 262 529642 62 kde-d(10)-kke262 529667 75 kddk-d(9)-kke 262 529692 53 kdde-d(9)-kke 262 529717 69eddk-d(9)-kke 262 529722 74 ke-d(10)-keke 262 529742 81 ek-d(10)-keke262 529762 66 ke-d(10)-kdke 262 529782 68 ek-d(10)-kdke 262 529851 68kde-d(10)-kdke 418 529871 77 edk-d(10)-kdke 418 529891 36k-(d4)-k-(d4)-k-(d4)-ke 418 529910 60 eeee-d(9)-kke 262 529568 79eek-d(10)-kke 263 529593 70 kee-d(10)-kke 263 529618 77 edk-d(10)-kke263 529643 72 kde-d(10)-kke 263 529668 73 kddk-d(9)-kke 263 529693 62kdde-d(9)-kke 263 529718 69 eddk-d(9)-kke 263 529911 66 eeee-d(9)-kke263 529462 76 eek-d(10)-keke 419 529268 18 eek-d(10)-keke 420 529187 46eek-d(10)-keke 421 529224 48 eek-d(10)-kkke 421 529130 34 eee-d(10)-kkk422 529150 51 kkk-d(10)-eee 422 529549 85 eek-d(10)-kke 42 529574 81kee-d(10)-kke 42 529599 64 edk-d(10)-kke 42 529624 68 kde-d(10)-kke 42529649 77 kddk-d(9)-kke 42 529674 65 kdde-d(9)-kke 42 529699 63eddk-d(9)-kke 42 529931 59 eeee-d(9)-kke 42 529810 80 k-d(10)-kekee 423529829 67 k-d(10)-kdkee 423 529269 65 eek-d(10)-keke 424 529731 66ke-d(10)-keke 425 529751 76 ek-d(10)-keke 425 529771 73 ke-d(10)-kdke425 529791 65 ek-d(10)-kdke 425 529860 73 kde-d(10)-kdke 424 529880 74edk-d(10)-kdke 424 529900 62 k-(d4)-k-(d4)-k-(d4)-ke 424 529270 69eek-d(10)-keke 480 529550 81 eek-d(10)-kke 44 529575 88 kee-d(10)-kke 44529600 78 edk-d(10)-kke 44 529625 74 kde-d(10)-kke 44 529650 81kddk-d(9)-kke 44 529675 76 kdde-d(9)-kke 44 529700 73 eddk-d(9)-kke 44529920 67 eeee-d(9)-kke 44 529271 43 eek-d(10)-keke 427 529272 0eek-d(10)-keke 428 529273 62 eek-d(10)-keke 429 529274 78 eek-d(10)-keke430 529275 70 eek-d(10)-keke 431 529276 73 eek-d(10)-keke 432 529277 71eek-d(10)-keke 433 529278 72 eek-d(10)-keke 434 529279 10 eek-d(10)-keke435 529280 11 eek-d(10)-keke 436 529281 82 eek-d(10)-keke 437 529282 87eek-d(10)-keke 438 529803 71 k-d(10)-kekee 250 529822 72 k-d(10)-kdkee250 529724 76 ke-d(10)-keke 439 529744 81 ek-d(10)-keke 439 529764 65ke-d(10)-kdke 439 529784 68 ek-d(10)-kdke 439 529853 64 kde-d(10)-kdke440 529873 69 edk-d(10)-kdke 440 529893 45 k-(d4)-k-(d4)-k-(d4)-ke 440529937 81 eek-d(10)-keke 440 529551 88 eek-d(10)-kke 48 529576 71kee-d(10)-kke 48 529601 74 edk-d(10)-kke 48 529626 72 kde-d(10)-kke 48529651 85 kddk-d(9)-kke 48 529676 67 kdde-d(9)-kke 48 529701 82eddk-d(9)-kke 48 529913 76 eeee-d(9)-kke 48 529811 56 k-d(10)-kekee 441529830 46 k-d(10)-kdkee 441 529732 63 ke-d(10)-keke 442 529752 72ek-d(10)-keke 442 529772 61 ke-d(10)-kdke 442 529792 68 ek-d(10)-kdke442 529861 54 kde-d(10)-kdke 443 529881 78 edk-d(10)-kdke 443 529901 29k-(d4)-k-(d4)-k-(d4)-ke 443 529939 67 eek-d(10)-keke 443 529283 70eek-d(10)-keke 444 529552 72 eek-d(10)-kke 49 529577 80 kee-d(10)-kke 49529602 64 edk-d(10)-kke 49 529627 56 kde-d(10)-kke 49 529652 57kddk-d(9)-kke 49 529677 43 kdde-d(9)-kke 49 529702 54 eddk-d(9)-kke 49529921 42 eeee-d(9)-kke 49 529284 76 eek-d(10)-keke 445 529285 77eek-d(10)-keke 446 529286 68 eek-d(10)-keke 447 529287 65 eek-d(10)-keke448 529719 73 ke-d(10)-keke 264 529739 83 ek-d(10)-keke 264 529759 63ke-d(10)-kdke 264 529779 70 ek-d(10)-kdke 244 529848 60 kde-d(10)-kdke449 529868 63 edk-d(10)-kdke 449 529888 53 k-(d4)-k-(d4)-k-(d4)-ke 449529553 81 eek-d(10)-kke 265 529578 65 kee-d(10)-kke 265 529603 60edk-d(10)-kke 265 529628 59 kde-d(10)-kke 265 529653 76 kddk-d(9)-kke265 529678 56 kdde-d(9)-kke 265 529703 68 eddk-d(9)-kke 265 529908 69eeee-d(9)-kke 265 529168 64 eek-d(10)-keke 450 529205 62 eek-d(10)-kkke450 529290 53 eek-d(10)-keke 451 529802 57 k-d(10)-kekee 452 529821 61k-d(10)-kdkee 452 529292 74 eek-d(10)-keke 453 529723 68 ke-d(10)-keke454 529743 84 ek-d(10)-keke 454 529763 64 ke-d(10)-kdke 454 529783 72ek-d(10)-kdke 454 529852 66 kde-d(10)-kdke 453 529872 62 edk-d(10)-kdke453 529892 43 k-(d4)-k-(d4)-k-(d4)-ke 453 529554 80 eek-d(10)-kke 252529579 83 kee-d(10)-kke 252 529604 73 edk-d(10)-kke 252 529629 64kde-d(10)-kke 252 529654 69 kddk-d(9)-kke 252 529679 52 kdde-d(9)-kke252 529704 63 eddk-d(9)-kke 252 529912 64 eeee-d(9)-kke 252 529294 74eek-d(10)-keke 455 529296 52 eek-d(10)-keke 456 529298 60 eek-d(10)-keke457 529300 71 eek-d(10)-keke 458 529188 79 eek-d(10)-keke 459 529225 78eek-d(10)-kkke 459 529131 58 eee-d(10)-kkk 460 529151 71 kkk-d(10)-eee460 529302 74 eek-d(10)-keke 461 529189 64 eek-d(10)-keke 222 529226 50eek-d(10)-kkke 222 529132 78 eee-d(10)-kkk 462 529152 62 kkk-d(10)-eee462 529190 76 eek-d(10)-keke 223 529227 88 eek-d(10)-kkke 250 529133 81eee-d(10)-kkk 463 529153 68 kkk-d(10)-eee 463 529191 78 eek-d(10)-keke224 529228 85 eek-d(10)-kkke 224 529134 75 eee-d(10)-kkk 464 529154 61kkk-d(10)-eee 464 529304 89 eek-d(10)-keke 465 529306 84 eek-d(10)-keke466 529308 68 eek-d(10)-keke 467 529310 59 eek-d(10)-keke 468 529169 79eek-d(10)-keke 469 529206 82 eek-d(10)-kkke 469 529312 68 eek-d(10)-keke470 529314 61 eek-d(10)-keke 471 529316 62 eek-d(10)-keke 472 529555 78eek-d(10)-kke 59 529580 73 kee-d(10)-kke 59 529605 71 edk-d(10)-kke 59529630 64 kde-d(10)-kke 59 529655 63 kddk-d(9)-kke 59 529680 43kdde-d(9)-kke 59 529705 63 eddk-d(9)-kke 59 529932 60 eeee-d(9)-kke 59529318 82 eek-d(10)-keke 473 529170 85 eek-d(10)-keke 474 529207 88eek-d(10)-kkke 474 529171 81 eek-d(10)-keke 475 529208 84 eek-d(10)-kkke475 529805 40 k-d(10)-kekee 476 529824 32 k-d(10)-kdkee 476 529320 74eek-d(10)-keke 477 529726 80 ke-d(10)-keke 478 529746 82 ek-d(10)-keke478 529766 63 ke-d(10)-kdke 478 529786 69 ek-d(10)-kdke 478 529855 39kde-d(10)-kdke 477 529875 40 edk-d(10)-kdke 477 529895 27k-(d4)-k-(d4)-k-(d4)-ke 477 529556 72 eek-d(10)-kke 61 529581 68kee-d(10)-kke 61 529606 54 edk-d(10)-kke 61 529631 29 kde-d(10)-kke 61529656 74 kddk-d(9)-kke 61 529681 32 kdde-d(9)-kke 61 529706 41eddk-d(9)-kke 61 529915 51 eeee-d(9)-kke 61 529172 88 eek-d(10)-keke 226529209 87 eek-d(10)-kkke 226 529173 92 eek-d(10)-keke 227 529210 89eek-d(10)-kkke 227 529183 85 eek-d(10)-keke 479 529220 92 eek-d(10)-kkke479 529126 83 eee-d(10)-kkk 257 529146 84 kkk-d(10)-eee 257 529174 85eek-d(10)-keke 480 529211 86 eek-d(10)-kkke 480 529322 71 eek-d(10)-keke481 529324 79 eek-d(10)-keke 482 529326 85 eek-d(10)-keke 483 529175 92eek-d(10)-keke 228 529212 92 eek-d(10)-kkke 228 529176 89 eek-d(10)-keke229 529213 90 eek-d(10)-kkke 229 529804 89 k-d(10)-kekee 259 529823 89k-d(10)-kdkee 259 529166 83 eek-d(10)-keke 230 529203 86 eek-d(10)-kkke230 529725 92 ke-d(10)-keke 260 529745 91 ek-d(10)-keke 260 529765 88ke-d(10)-kdke 260 529785 91 ek-d(10)-kdke 260 529799 89 k-d(10)-kekee260 529818 88 k-d(10)-kdkee 260 529854 90 kde-d(10)-kdke 230 529874 81edk-d(10)-kdke 230 529894 60 k-(d4)-k-(d4)-k-(d4)-ke 230 529167 71eek-d(10)-keke 231 529204 70 eek-d(10)-kkke 231 529557 86 eek-d(10)-kke69 529582 86 kee-d(10)-kke 69 529607 84 edk-d(10)-kke 69 529632 81kde-d(10)-kke 69 529657 85 kddk-d(9)-kke 69 529682 78 kdde-d(9)-kke 69529707 79 eddk-d(9)-kke 69 529720 75 ke-d(10)-keke 69 529740 70ek-d(10)-keke 69 529760 78 ke-d(10)-kdke 69 529780 83 ek-d(10)-kdke 69529849 80 kde-d(10)-kdke 231 529869 72 edk-d(10)-kdke 231 529889 49k-(d4)-k-(d4)-k-(d4)-ke 231 529914 69 eeee-d(9)-kke 69 529328 68eek-d(10)-keke 484 529558 71 eek-d(10)-kke 71 529583 81 kee-d(10)-kke 71529608 68 edk-d(10)-kke 71 529633 73 kde-d(10)-kke 71 529658 63kddk-d(9)-kke 71 529683 74 kdde-d(9)-kke 71 529708 70 eddk-d(9)-kke 71529909 59 eeee-d(9)-kke 71 529192 51 eek-d(10)-keke 485 529229 69eek-d(10)-kkke 485 529135 54 eee-d(10)-kkk 486 529155 56 kkk-d(10)-eee486 529330 37 eek-d(10)-keke 487 e = 2′ -MOE, k = cEt, d =2′-deoxyribonucleoside

Example 20 Design of Modified Oligonucleotides Comprising2′-O-Methoxyethyl (2′-MOE) or 6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt)Modifications

Based on the activity of the antisense oligonucleotides listed above,additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid targeting start positions 1147, 1154 or 12842 of Target-X.

The newly designed chimeric antisense oligonucleotides are 16 or 17nucleotides in length and their motifs are described in Table 35. Thechemistry column of Table 35 presents the sugar motif of eacholigonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE)nucleoside, “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g cEt) and“d” indicates a 2′-deoxyribonucleoside. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytosineresidues throughout each oligonucleotide are 5-methylcytosine.

Each gapmer listed in Table 35 is targeted to the human Target-X genomicsequence.

TABLE 35 Chimeric antisense oligonucleotides targeted to Target-X ISISNo Chemistry SEQ CODE 529544 eek-d(10)-kke 21 529569 kee-d(10)-kke 21529594 edk-d(10)-kke 21 529619 kde-d(10)-kke 21 529644 kddk-d(9)-kke 21529669 kdde-d(9)-kke 21 529694 eddk-d(9)-kke 21 529929 eeee-d(9)-kke 21529809 k-d(10)-kekee 488 529828 k-d(10)-kdkee 488 529730 ke-d(10)-keke489 529750 ek-d(10)-keke 489 529770 ke-d(10)-kdke 489 529790ek-d(10)-kdke 489 529859 kde-d(10)-kdke 490 529879 edk-d(10)-kdke 490529899 k-d(4)-k-d(4)-k-d(4)-ke 490 529545 eek-d(10)-kke 22 529570kee-d(10)-kke 22 529595 edk-d(10)-kke 22 529620 kde-d(10)-kke 22 529645kddk-d(9)-kke 22 529670 kdde-d(9)-kke 22 529695 eddk-d(9)-kke 22 529919eeee-d(9)-kke 22 529548 eek-d(10)-kke 41 529573 kee-d(10)-kke 41 529598edk-d(10)-kke 41 529623 kde-d(10)-kke 41 529648 kddk-d(9)-kke 41 529673kdde-d(9)-kke 41 529698 eddk-d(9)-kke 41 529930 eeee-d(9)-kke 41 e =2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 21 Modified Oligonucleotides Comprising 2′-O-Methoxyethyl(2′-MOE) and 6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt) ModificationsTargeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-Xnucleic acid and were tested for their effects on Target-X mRNA invitro. ISIS 472998 and ISIS 515554, described in the Examples above werealso included in the screen.

The newly designed chimeric antisense oligonucleotides are 16nucleotides in length and their motifs are described in Table 36. Thechemistry column of Table 36 presents the sugar motif of eacholigonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE)nucleoside, “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g cEt) and“d” indicates a 2′-deoxyribonucleoside. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytosineresidues throughout each oligonucleotide are 5-methylcytosine.

Each gapmer listed in Table 36 is targeted to the human Target-X genomicsequence.

Cultured Hep3B cells at a density of 20,000 cells per well weretransfected using electroporation with 2,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and Target-X mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS2927 was used tomeasure mRNA levels. Target-X mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of Target-X, relative to untreated control cells.

TABLE 36 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X ISIS No % inhibition Chemistry SEQCODE 472998 88 kk-d(10)-kk 74 515554 75 eee-d(10)-kkk 493 534530 92keke-d(9)-kek 491 534563 92 kek-d(9)-ekek 491 534596 88 ekee-d(9)-kke491 534629 89 eke-d(9)-ekke 491 534662 87 eekk-d(9)-eke 491 534695 92eek-d(9)-keke 491 534732 90 ekek-d(8)-keke 491 534767 92 keek-d(8)-keek491 534802 93 ekk-d(10)-kke 491 534832 83 edk-d(10)-kke 491 534862 72kde-d(10)-kke 491 534892 82 eek-d(10)-kke 491 534922 80 kddk-d(9)-kke491 534952 72 kdde-d(9)-kke 491 534982 77 eddk-d(9)-kke 491 535012 70eeee-d(9)-kke 491 535045 84 eeee-d(9)-kkk 491 535078 87 eeek-d(9)-kke491 535111 63 eeeee-d(8)-kke 491 535144 69 ededk-d(8)-kke 491 535177 68edkde-d(8)-kke 491 534531 61 keke-d(9)-kek 492 534564 30 kek-d(9)-ekek492 534597 67 ekee-d(9)-kke 492 534630 54 eke-d(9)-ekke 492 534663 94eekk-d(9)-eke 492 534696 68 eek-d(9)-keke 492 534733 44 ekek-d(8)-keke492 534768 55 keek-d(8)-keek 492 534803 73 ekk-d(10)-kke 492 534833 65edk-d(10)-kke 492 534863 53 kde-d(10)-kke 492 534893 61 eek-d(10)-kke492 534923 70 kddk-d(9)-kke 492 534953 54 kdde-d(9)-kke 492 534983 58eddk-d(9)-kke 492 535013 52 eeee-d(9)-kke 492 535046 67 eeee-d(9)-kkk492 535079 57 eeek-d(9)-kke 492 535112 42 eeeee-d(8)-kke 492 535145 41ededk-d(8)-kke 492 535178 35 edkde-d(8)-kke 492 534565 87 kek-d(9)-ekek493 534598 72 ekee-d(9)-kke 493 534631 70 eke-d(9)-ekke 493 534664 94eekk-d(9)-eke 493 534697 90 eek-d(9)-keke 493 534734 74 ekek-d(8)-keke493 534769 80 keek-d(8)-keek 493 534804 87 ekk-d(10)-kke 493 534834 76edk-d(10)-kke 493 534864 56 kde-d(10)-kke 493 534894 67 eek-d(10)-kke493 534924 71 kddk-d(9)-kke 493 534954 54 kdde-d(9)-kke 493 534984 48eddk-d(9)-kke 493 535014 43 eeee-d(9)-kke 493 535047 60 eeee-d(9)-kkk493 535080 64 eeek-d(9)-kke 493 535113 32 eeeee-d(8)-kke 493 535146 31ededk-d(8)-kke 493 535179 28 edkde-d(8)-kke 493 534533 82 keke-d(9)-kek494 534566 88 kek-d(9)-ekek 494 534599 65 ekee-d(9)-kke 494 534632 69eke-d(9)-ekke 494 534665 87 eekk-d(9)-eke 494 534698 64 eek-d(9)-keke494 534735 63 ekek-d(8)-keke 494 534770 66 keek-d(8)-keek 494 534805 87ekk-d(10)-kke 494 534835 68 edk-d(10)-kke 494 534865 66 kde-d(10)-kke494 534895 57 eek-d(10)-kke 494 534925 82 kddk-d(9)-kke 494 534955 76kdde-d(9)-kke 494 534985 71 eddk-d(9)-kke 494 535015 59 eeee-d(9)-kke494 535048 69 eeee-d(9)-kkk 494 535081 67 eeek-d(9)-kke 494 535114 37eeeee-d(8)-kke 494 535147 32 ededk-d(8)-kke 494 535180 31 edkde-d(8)-kke494 534534 94 keke-d(9)-kek 234 534567 92 kek-d(9)-ekek 234 534600 92ekee-d(9)-kke 234 534633 91 eke-d(9)-ekke 234 534666 89 eekk-d(9)-eke234 534699 91 eek-d(9)-keke 234 534736 83 ekek-d(8)-keke 234 534771 80keek-d(8)-keek 234 534806 96 ekk-d(10)-kke 234 534836 86 edk-d(10)-kke234 534866 82 kde-d(10)-kke 234 534896 82 eek-d(10)-kke 234 534926 89kddk-d(9)-kke 234 534956 91 kdde-d(9)-kke 234 534986 87 eddk-d(9)-kke234 535016 83 eeee-d(9)-kke 234 535049 87 eeee-d(9)-kkk 234 535082 87eeek-d(9)-kke 234 535115 77 eeeee-d(8)-kke 234 535148 73 ededk-d(8)-kke234 535181 68 edkde-d(8)-kke 234 534535 66 keke-d(9)-kek 236 534568 85kek-d(9)-ekek 236 534601 51 ekee-d(9)-kke 236 534634 80 eke-d(9)-ekke236 534667 90 eekk-d(9)-eke 236 534700 88 eek-d(9)-keke 236 534737 65ekek-d(8)-keke 236 534772 77 keek-d(8)-keek 236 534807 84 ekk-d(10)-kke236 534837 78 edk-d(10)-kke 236 534867 44 kde-d(10)-kke 236 534897 82eek-d(10)-kke 236 534927 61 kddk-d(9)-kke 236 534957 58 kdde-d(9)-kke236 534987 49 eddk-d(9)-kke 236 535017 38 eeee-d(9)-kke 236 535050 32eeee-d(9)-kkk 236 535083 43 eeek-d(9)-kke 236 535116 9 eeeee-d(8)-kke236 535149 23 ededk-d(8)-kke 236 535182 18 edkde-d(8)-kke 236 534536 89keke-d(9)-kek 238 534569 90 kek-d(9)-ekek 238 534602 85 ekee-d(9)-kke238 534635 87 eke-d(9)-ekke 238 534668 90 eekk-d(9)-eke 238 534701 92eek-d(9)-keke 238 534738 81 ekek-d(8)-keke 238 534773 79 keek-d(8)-keek238 534808 90 ekk-d(10)-kke 238 534838 88 edk-d(10)-kke 238 534868 67kde-d(10)-kke 238 534898 89 eek-d(10)-kke 238 534928 81 kddk-d(9)-kke238 534958 78 kdde-d(9)-kke 238 534988 66 eddk-d(9)-kke 238 535018 78eeee-d(9)-kke 238 535051 76 eeee-d(9)-kkk 238 535084 80 eeek-d(9)-kke238 535117 58 eeeee-d(8)-kke 238 535150 51 ededk-d(8)-kke 238 535183 53edkde-d(8)-kke 238 534537 91 keke-d(9)-kek 239 534570 85 kek-d(9)-ekek239 534603 79 ekee-d(9)-kke 239 534636 72 eke-d(9)-ekke 239 534669 85eekk-d(9)-eke 239 534702 85 eek-d(9)-keke 239 534739 73 ekek-d(8)-keke239 534774 77 keek-d(8)-keek 239 534809 91 ekk-d(10)-kke 239 534839 86edk-d(10)-kke 239 534869 71 kde-d(10)-kke 239 534899 82 eek-d(10)-kke239 534929 83 kddk-d(9)-kke 239 534959 80 kdde-d(9)-kke 239 534989 79eddk-d(9)-kke 239 535019 76 eeee-d(9)-kke 239 535052 79 eeee-d(9)-kkk239 535085 81 eeek-d(9)-kke 239 535118 58 eeeee-d(8)-kke 239 535151 65ededk-d(8)-kke 239 535184 60 edkde-d(8)-kke 239 534516 77 keke-d(9)-kek495 534549 80 kek-d(9)-ekek 495 534582 73 ekee-d(9)-kke 495 534615 79eke-d(9)-ekke 495 534648 67 eekk-d(9)-eke 495 534681 87 eek-d(9)-keke495 534718 46 ekek-d(8)-keke 495 534753 68 keek-d(8)-keek 495 534788 84ekk-d(10)-kke 495 534818 82 edk-d(10)-kke 495 534848 75 kde-d(10)-kke495 534878 72 eek-d(10)-kke 495 534908 81 kddk-d(9)-kke 495 534938 69kdde-d(9)-kke 495 534968 77 eddk-d(9)-kke 495 534998 76 eeee-d(9)-kke495 535031 76 eeee-d(9)-kkk 495 535064 70 eeek-d(9)-kke 495 535097 57eeeee-d(8)-kke 495 535130 69 ededk-d(8)-kke 495 535163 58 edkde-d(8)-kke495 534538 71 keke-d(9)-kek 241 534571 64 kek-d(9)-ekek 241 534604 66ekee-d(9)-kke 241 534637 74 eke-d(9)-ekke 241 534670 87 eekk-d(9)-eke241 534703 72 eek-d(9)-keke 241 534740 56 ekek-d(8)-keke 241 534775 53keek-d(8)-keek 241 534810 78 ekk-d(10)-kke 241 534840 73 edk-d(10)-kke241 534870 65 kde-d(10)-kke 241 534900 69 eek-d(10)-kke 241 534930 67kddk-d(9)-kke 241 534960 62 kdde-d(9)-kke 241 534990 66 eddk-d(9)-kke241 535020 61 eeee-d(9)-kke 241 535053 47 eeee-d(9)-kkk 241 535086 61eeek-d(9)-kke 241 535119 49 eeeee-d(8)-kke 241 535152 48 ededk-d(8)-kke241 535185 57 edkde-d(8)-kke 241 534539 70 keke-d(9)-kek 496 534572 82kek-d(9)-ekek 496 534605 59 ekee-d(9)-kke 496 534638 69 eke-d(9)-ekke496 534671 89 eekk-d(9)-eke 496 534704 83 eek-d(9)-keke 496 534741 47ekek-d(8)-keke 496 534776 46 keek-d(8)-keek 496 534811 71 ekk-d(10)-kke496 534841 61 edk-d(10)-kke 496 534871 53 kde-d(10)-kke 496 534901 55eek-d(10)-kke 496 534931 73 kddk-d(9)-kke 496 534961 53 kdde-d(9)-kke496 534991 56 eddk-d(9)-kke 496 535021 58 eeee-d(9)-kke 496 535054 59eeee-d(9)-kkk 496 535087 0 eeek-d(9)-kke 496 535120 41 eeeee-d(8)-kke496 535153 44 ededk-d(8)-kke 496 535186 35 edkde-d(8)-kke 496 534573 76kek-d(9)-ekek 497 534606 55 ekee-d(9)-kke 497 534639 72 eke-d(9)-ekke497 534672 89 eekk-d(9)-eke 497 534705 87 eek-d(9)-keke 497 534742 84ekek-d(8)-keke 497 534777 79 keek-d(8)-keek 497 534812 76 ekk-d(10)-kke497 534842 74 edk-d(10)-kke 497 534872 53 kde-d(10)-kke 497 534902 70eek-d(10)-kke 497 534932 73 kddk-d(9)-kke 497 534962 60 kdde-d(9)-kke497 534992 61 eddk-d(9)-kke 497 535022 38 eeee-d(9)-kke 497 535055 42eeee-d(9)-kkk 497 535088 56 eeek-d(9)-kke 497 535121 5 eeeee-d(8)-kke497 535154 22 ededk-d(8)-kke 497 535187 16 edkde-d(8)-kke 497 534541 86keke-d(9)-kek 498 534574 89 kek-d(9)-ekek 498 534607 59 ekee-d(9)-kke498 534640 76 eke-d(9)-ekke 498 534673 89 eekk-d(9)-eke 498 534706 86eek-d(9)-keke 498 534743 79 ekek-d(8)-keke 498 534778 80 keek-d(8)-keek498 534813 83 ekk-d(10)-kke 498 534843 82 edk-d(10)-kke 498 534873 83kde-d(10)-kke 498 534903 78 eek-d(10)-kke 498 534933 83 kddk-d(9)-kke498 534963 70 kdde-d(9)-kke 498 534993 78 eddk-d(9)-kke 498 535023 56eeee-d(9)-kke 498 535056 59 eeee-d(9)-kkk 498 535089 73 eeek-d(9)-kke498 535122 39 eeeee-d(8)-kke 498 535155 60 ededk-d(8)-kke 498 535188 41edkde-d(8)-kke 498 534542 75 keke-d(9)-kek 499 534575 82 kek-d(9)-ekek499 534608 72 ekee-d(9)-kke 499 534641 69 eke-d(9)-ekke 499 534674 84eekk-d(9)-eke 499 534707 78 eek-d(9)-keke 499 534744 72 ekek-d(8)-keke499 534779 75 keek-d(8)-keek 499 534814 81 ekk-d(10)-kke 499 534844 75edk-d(10)-kke 499 534874 70 kde-d(10)-kke 499 534904 71 eek-d(10)-kke499 534934 73 kddk-d(9)-kke 499 534964 72 kdde-d(9)-kke 499 534994 69eddk-d(9)-kke 499 535024 56 eeee-d(9)-kke 499 535057 63 eeee-d(9)-kkk499 535090 64 eeek-d(9)-kke 499 535123 40 eeeee-d(8)-kke 499 535156 47ededk-d(8)-kke 499 535189 48 edkde-d(8)-kke 499 534515 52 keke-d(9)-kek34 534548 85 kek-d(9)-ekek 34 534581 75 ekee-d(9)-kke 34 534614 83eke-d(9)-ekke 34 534647 65 eekk-d(9)-eke 34 534680 88 eek-d(9)-keke 34534717 76 ekek-d(8)-keke 34 534752 79 keek-d(8)-keek 34 534787 90ekk-d(10)-kke 34 535030 77 eeee-d(9)-kkk 34 535063 75 eeek-d(9)-kke 34535096 54 eeeee-d(8)-kke 34 535129 66 ededk-d(8)-kke 34 535162 49edkde-d(8)-kke 34 534543 66 keke-d(9)-kek 500 534576 69 kek-d(9)-ekek500 534609 77 ekee-d(9)-kke 500 534642 62 eke-d(9)-ekke 500 534675 80eekk-d(9)-eke 500 534708 81 eek-d(9)-keke 500 534745 68 ekek-d(8)-keke500 534780 69 keek-d(8)-keek 500 534815 85 ekk-d(10)-kke 500 534845 72edk-d(10)-kke 500 534875 56 kde-d(10)-kke 500 534905 65 eek-d(10)-kke500 534935 78 kddk-d(9)-kke 500 534965 48 kdde-d(9)-kke 500 534995 62eddk-d(9)-kke 500 535025 58 eeee-d(9)-kke 500 535058 60 eeee-d(9)-kkk500 535091 61 eeek-d(9)-kke 500 535124 51 eeeee-d(8)-kke 500 535157 55ededk-d(8)-kke 500 535190 47 edkde-d(8)-kke 500 534517 71 keke-d(9)-kek501 534550 80 kek-d(9)-ekek 501 534583 70 ekee-d(9)-kke 501 534616 84eke-d(9)-ekke 501 534649 68 eekk-d(9)-eke 501 534682 87 eek-d(9)-keke501 534719 90 ekek-d(8)-keke 501 534754 83 keek-d(8)-keek 501 534789 86ekk-d(10)-kke 501 534819 69 edk-d(10)-kke 501 534849 62 kde-d(10)-kke501 534879 69 eek-d(10)-kke 501 534909 73 kddk-d(9)-kke 501 534939 49kdde-d(9)-kke 501 534969 47 eddk-d(9)-kke 501 534999 51 eeee-d(9)-kke501 535032 51 eeee-d(9)-kkk 501 535065 64 eeek-d(9)-kke 501 535098 31eeeee-d(8)-kke 501 535131 31 ededk-d(8)-kke 501 535164 40 edkde-d(8)-kke501 534518 81 keke-d(9)-kek 502 534551 88 kek-d(9)-ekek 502 534584 78ekee-d(9)-kke 502 534617 80 eke-d(9)-ekke 502 534650 83 eekk-d(9)-eke502 534683 93 eek-d(9)-keke 502 534720 87 ekek-d(8)-keke 502 534755 82keek-d(8)-keek 502 534790 89 ekk-d(10)-kke 502 534820 64 edk-d(10)-kke502 534850 38 kde-d(10)-kke 502 534880 68 eek-d(10)-kke 502 534910 60kddk-d(9)-kke 502 534940 37 kdde-d(9)-kke 502 534970 59 eddk-d(9)-kke502 535000 30 eeee-d(9)-kke 502 535033 44 eeee-d(9)-kkk 502 535066 64eeek-d(9)-kke 502 535099 22 eeeee-d(8)-kke 502 535132 54 ededk-d(8)-kke502 535165 45 edkde-d(8)-kke 502 534544 80 keke-d(9)-kek 503 534577 83kek-d(9)-ekek 503 534610 62 ekee-d(9)-kke 503 534643 66 eke-d(9)-ekke503 534676 95 eekk-d(9)-eke 503 534709 86 eek-d(9)-keke 503 534746 73ekek-d(8)-keke 503 534781 71 keek-d(8)-keek 503 534816 83 ekk-d(10)-kke503 534846 73 edk-d(10)-kke 503 534876 39 kde-d(10)-kke 503 534906 67eek-d(10)-kke 503 534936 66 kddk-d(9)-kke 503 534966 48 kdde-d(9)-kke503 534996 56 eddk-d(9)-kke 503 535026 39 eeee-d(9)-kke 503 535059 45eeee-d(9)-kkk 503 535092 48 eeek-d(9)-kke 503 535125 26 eeeee-d(8)-kke503 535158 44 ededk-d(8)-kke 503 535191 34 edkde-d(8)-kke 503 534545 83keke-d(9)-kek 504 534578 81 kek-d(9)-ekek 504 534611 78 ekee-d(9)-kke504 534644 72 eke-d(9)-ekke 504 534677 92 eekk-d(9)-eke 504 534710 78eek-d(9)-keke 504 534747 85 ekek-d(8)-keke 504 534782 85 keek-d(8)-keek504 534817 88 ekk-d(10)-kke 504 534847 73 edk-d(10)-kke 504 534877 66kde-d(10)-kke 504 534907 73 eek-d(10)-kke 504 534937 85 kddk-d(9)-kke504 534967 80 kdde-d(9)-kke 504 534997 74 eddk-d(9)-kke 504 535027 64eeee-d(9)-kke 504 535060 68 eeee-d(9)-kkk 504 535093 73 eeek-d(9)-kke504 535126 42 eeeee-d(8)-kke 504 535159 49 ededk-d(8)-kke 504 535192 51edkde-d(8)-kke 504 534519 87 keke-d(9)-kek 505 534552 85 kek-d(9)-ekek505 534585 76 ekee-d(9)-kke 505 534618 78 eke-d(9)-ekke 505 534651 79eekk-d(9)-eke 505 534684 87 eek-d(9)-keke 505 534721 89 ekek-d(8)-keke505 534756 90 keek-d(8)-keek 505 534791 84 ekk-d(10)-kke 505 534821 79edk-d(10)-kke 505 534851 64 kde-d(10)-kke 505 534881 65 eek-d(10)-kke505 534911 85 kddk-d(9)-kke 505 534941 66 kdde-d(9)-kke 505 534971 75eddk-d(9)-kke 505 535001 62 eeee-d(9)-kke 505 535034 65 eeee-d(9)-kkk505 535067 76 eeek-d(9)-kke 505 535100 5 eeeee-d(8)-kke 505 535133 30ededk-d(8)-kke 505 535166 23 edkde-d(8)-kke 505 534520 87 keke-d(9)-kek251 534553 79 kek-d(9)-ekek 251 534586 60 ekee-d(9)-kke 251 534619 62eke-d(9)-ekke 251 534652 84 eekk-d(9)-eke 251 534685 84 eek-d(9)-keke251 534722 75 ekek-d(8)-keke 251 534757 81 keek-d(8)-keek 251 534792 87ekk-d(10)-kke 251 534822 80 edk-d(10)-kke 251 534852 38 kde-d(10)-kke251 534882 75 eek-d(10)-kke 251 534912 74 kddk-d(9)-kke 251 534942 58kdde-d(9)-kke 251 534972 59 eddk-d(9)-kke 251 535002 50 eeee-d(9)-kke251 535035 57 eeee-d(9)-kkk 251 535068 67 eeek-d(9)-kke 251 535101 24eeeee-d(8)-kke 251 535134 23 ededk-d(8)-kke 251 535167 26 edkde-d(8)-kke251 534513 90 keke-d(9)-kek 252 534546 92 kek-d(9)-ekek 252 534579 78ekee-d(9)-kke 252 534612 82 eke-d(9)-ekke 252 534645 73 eekk-d(9)-eke252 534678 91 eek-d(9)-keke 252 534715 87 ekek-d(8)-keke 252 534750 88keek-d(8)-keek 252 534785 89 ekk-d(10)-kke 252 535028 52 eeee-d(9)-kkk252 535061 73 eeek-d(9)-kke 252 535094 61 eeeee-d(8)-kke 252 535127 59ededk-d(8)-kke 252 535160 62 edkde-d(8)-kke 252 534521 86 keke-d(9)-kek506 534554 87 kek-d(9)-ekek 506 534587 62 ekee-d(9)-kke 506 534620 68eke-d(9)-ekke 506 534653 77 eekk-d(9)-eke 506 534686 90 eek-d(9)-keke506 534723 88 ekek-d(8)-keke 506 534758 79 keek-d(8)-keek 506 534793 85ekk-d(10)-kke 506 534823 81 edk-d(10)-kke 506 534853 59 kde-d(10)-kke506 534883 69 eek-d(10)-kke 506 534913 76 kddk-d(9)-kke 506 534943 53kdde-d(9)-kke 506 534973 61 eddk-d(9)-kke 506 535003 53 eeee-d(9)-kke506 535036 35 eeee-d(9)-kkk 506 535069 62 eeek-d(9)-kke 506 535102 31eeeee-d(8)-kke 506 535135 44 ededk-d(8)-kke 506 535168 34 edkde-d(8)-kke506 534522 83 keke-d(9)-kek 507 534555 81 kek-d(9)-ekek 507 534588 72ekee-d(9)-kke 507 534621 74 eke-d(9)-ekke 507 534654 78 eekk-d(9)-eke507 534687 91 eek-d(9)-keke 507 534724 84 ekek-d(8)-keke 507 534759 86keek-d(8)-keek 507 534794 78 ekk-d(10)-kke 507 534824 75 edk-d(10)-kke507 534854 63 kde-d(10)-kke 507 534884 60 eek-d(10)-kke 507 534914 75kddk-d(9)-kke 507 534944 69 kdde-d(9)-kke 507 534974 66 eddk-d(9)-kke507 535004 56 eeee-d(9)-kke 507 535037 50 eeee-d(9)-kkk 507 535070 68eeek-d(9)-kke 507 535103 55 eeeee-d(8)-kke 507 535136 51 ededk-d(8)-kke507 535169 54 edkde-d(8)-kke 507 534523 89 keke-d(9)-kek 253 534556 91kek-d(9)-ekek 253 534589 88 ekee-d(9)-kke 253 534622 93 eke-d(9)-ekke253 534655 72 eekk-d(9)-eke 253 534688 92 eek-d(9)-keke 253 534725 87ekek-d(8)-keke 253 534760 92 keek-d(8)-keek 253 534795 93 ekk-d(10)-kke253 534825 82 edk-d(10)-kke 253 534855 73 kde-d(10)-kke 253 534885 82eek-d(10)-kke 253 534915 88 kddk-d(9)-kke 253 534945 82 kdde-d(9)-kke253 534975 68 eddk-d(9)-kke 253 535005 69 eeee-d(9)-kke 253 535038 72eeee-d(9)-kkk 253 535071 74 eeek-d(9)-kke 253 535104 61 eeeee-d(8)-kke253 535137 67 ededk-d(8)-kke 253 535170 51 edkde-d(8)-kke 253 534524 95keke-d(9)-kek 254 534557 98 kek-d(9)-ekek 254 534590 91 ekee-d(9)-kke254 534623 91 eke-d(9)-ekke 254 534656 90 eekk-d(9)-eke 254 534689 92eek-d(9)-keke 254 534726 57 ekek-d(8)-keke 254 534761 89 keek-d(8)-keek254 534796 93 ekk-d(10)-kke 254 534826 89 edk-d(10)-kke 254 534856 87kde-d(10)-kke 254 534886 85 eek-d(10)-kke 254 534916 87 kddk-d(9)-kke254 534946 86 kdde-d(9)-kke 254 534976 77 eddk-d(9)-kke 254 535006 83eeee-d(9)-kke 254 535039 86 eeee-d(9)-kkk 254 535072 87 eeek-d(9)-kke254 535105 68 eeeee-d(8)-kke 254 535138 70 ededk-d(8)-kke 254 535171 65edkde-d(8)-kke 254 534558 92 kek-d(9)-ekek 255 534591 91 ekee-d(9)-kke255 534624 86 eke-d(9)-ekke 255 534657 90 eekk-d(9)-eke 255 534690 76eek-d(9)-keke 255 534727 92 ekek-d(8)-keke 255 534762 91 keek-d(8)-keek255 534797 94 ekk-d(10)-kke 255 534827 90 edk-d(10)-kke 255 534857 80kde-d(10)-kke 255 534887 76 eek-d(10)-kke 255 534917 91 kddk-d(9)-kke255 534947 91 kdde-d(9)-kke 255 534977 86 eddk-d(9)-kke 255 535007 80eeee-d(9)-kke 255 535040 86 eeee-d(9)-kkk 255 535073 87 eeek-d(9)-kke255 535106 70 eeeee-d(8)-kke 255 535139 73 ededk-d(8)-kke 255 535172 69edkde-d(8)-kke 255 534514 90 keke-d(9)-kek 61 534547 92 kek-d(9)-ekek 61534580 78 ekee-d(9)-kke 61 534613 80 eke-d(9)-ekke 61 534646 79eekk-d(9)-eke 61 534679 93 eek-d(9)-keke 61 534716 94 ekek-d(8)-keke 61534751 86 keek-d(8)-keek 61 534786 83 ekk-d(10)-kke 61 535029 45eeee-d(9)-kkk 61 535062 81 eeek-d(9)-kke 61 535095 57 eeeee-d(8)-kke 61535128 58 ededk-d(8)-kke 61 535161 49 edkde-d(8)-kke 61 534526 94keke-d(9)-kek 256 534559 95 kek-d(9)-ekek 256 534592 93 ekee-d(9)-kke256 534625 93 eke-d(9)-ekke 256 534658 93 eekk-d(9)-eke 256 534691 96eek-d(9)-keke 256 534728 93 ekek-d(8)-keke 256 534763 93 keek-d(8)-keek256 534798 97 ekk-d(10)-kke 256 534828 94 edk-d(10)-kke 256 534858 92kde-d(10)-kke 256 534888 93 eek-d(10)-kke 256 534918 95 kddk-d(9)-kke256 534948 93 kdde-d(9)-kke 256 534978 91 eddk-d(9)-kke 256 535008 88eeee-d(9)-kke 256 535041 87 eeee-d(9)-kkk 256 535074 90 eeek-d(9)-kke256 535107 78 eeeee-d(8)-kke 256 535140 81 ededk-d(8)-kke 256 535173 81edkde-d(8)-kke 256 534527 95 keke-d(9)-kek 258 534560 96 kek-d(9)-ekek258 534593 87 ekee-d(9)-kke 258 534626 85 eke-d(9)-ekke 258 534659 90eekk-d(9)-eke 258 534692 91 eek-d(9)-keke 258 534729 91 ekek-d(8)-keke258 534764 91 keek-d(8)-keek 258 534799 96 ekk-d(10)-kke 258 534829 91edk-d(10)-kke 258 534859 87 kde-d(10)-kke 258 534889 81 eek-d(10)-kke258 534919 92 kddk-d(9)-kke 258 534949 91 kdde-d(9)-kke 258 534979 84eddk-d(9)-kke 258 535009 78 eeee-d(9)-kke 258 535042 76 eeee-d(9)-kkk258 535075 83 eeek-d(9)-kke 258 535108 64 eeeee-d(8)-kke 258 535141 69ededk-d(8)-kke 258 535174 65 edkde-d(8)-kke 258 534528 94 keke-d(9)-kek260 534561 0 kek-d(9)-ekek 260 534594 92 ekee-d(9)-kke 260 534627 90eke-d(9)-ekke 260 534660 92 eekk-d(9)-eke 260 534693 95 eek-d(9)-keke260 534730 93 ekek-d(8)-keke 260 534765 92 keek-d(8)-keek 260 534800 93ekk-d(10)-kke 260 534830 93 edk-d(10)-kke 260 534860 85 kde-d(10)-kke260 534890 91 eek-d(10)-kke 260 534920 93 kddk-d(9)-kke 260 534950 90kdde-d(9)-kke 260 534980 88 eddk-d(9)-kke 260 535010 88 eeee-d(9)-kke260 535043 89 eeee-d(9)-kkk 260 535076 88 eeek-d(9)-kke 260 535109 76eeeee-d(8)-kke 260 535142 86 ededk-d(8)-kke 260 535175 71 edkde-d(8)-kke260 534529 70 keke-d(9)-kek 261 534562 86 kek-d(9)-ekek 261 534595 56ekee-d(9)-kke 261 534628 73 eke-d(9)-ekke 261 534661 64 eekk-d(9)-eke261 534694 75 eek-d(9)-keke 261 534731 47 ekek-d(8)-keke 261 534766 30keek-d(8)-keek 261 534801 83 ekk-d(10)-kke 261 534831 84 edk-d(10)-kke261 534861 71 kde-d(10)-kke 261 534891 73 eek-d(10)-kke 261 534921 55kddk-d(9)-kke 261 534951 61 kdde-d(9)-kke 261 534981 48 eddk-d(9)-kke261 535011 54 eeee-d(9)-kke 261 535044 46 eeee-d(9)-kkk 261 535077 29eeek-d(9)-kke 261 535110 19 eeeee-d(8)-kke 261 535143 15 ededk-d(8)-kke261 535176 37 edkde-d(8)-kke 261 e = 2′-MOE, k = cEt, d =2′-deoxynucleoside

Example 22 Modified Antisense Oligonucleotides Comprising2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH₃Bicyclic Nucleoside (e.g cEt)Modifications Targeting Human Target-X Targeting Intronic Repeats

Additional antisense oligonucleotides were designed targeting theintronic repeat regions of Target-X.

The newly designed chimeric antisense oligonucleotides and their motifsare described in Table 37. The internucleoside linkages throughout eachgapmer are phosphorothioate linkages (P═S) and are designated as “s”.Nucleosides followed by “d” indicate 2′-deoxyribonucleosides.Nucleosides followed by “k” indicate 6′-(S)—CH₃ bicyclic nucleosides(e.g cEt). Nucleosides followed by “e” indicate 2′-O-methoxyethyl(2′-MOE) nucleosides. “N” indicates modified or naturally occurringnucleobases (A, T, C, G, U, or 5-methyl C).

Each gapmer listed in Table 37 is targeted to the intronic region ofhuman Target-X genomic sequence, designated herein as Target-X.

Cultured Hep3B cells at a density of 20,000 cells per well weretransfected using electroporation with 2,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and Target-X mRNA levels were measured byquantitative real-time PCR. Human primer probe set was used to measuremRNA levels. Target-X mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of Target-X, relative to untreated control cells.

TABLE 37 Inhibition of human Target-X mRNA levels by chimeric antisenseoligonucleotides targeted to Target-X SEQ ISIS % SEQ ID Sequence (5′to 3′) No inhibition CODE NO Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds472998 90 508 7 Nds Nks Nk Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds473327 88  30 6 Nds Nds Nes Nes NeNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537024 74 509 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537025 79510 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537026 76 511 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537028 37 512 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537029 45513 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537030 67 514 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537031 59 515 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537032  9516 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537033 65 517 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537034 71 518 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537035 68519 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537036 74 520 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537038 69 521 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537039 67522 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537040 68 523 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537041 76 524 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537042 77525 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537043 70 526 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537044 82 527 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537045 69528 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537047 35 529 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537049 62 530 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537051 62531 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537055 16 532 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537056 25 533 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537057 49534 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537058 49 535 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537059 53 536 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537060 73537 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537061 70 538 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537062 69 539 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537063 68540 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537064 71 541 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537065 67 542 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537066 68543 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537067 71 544 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537068 86 545 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537069 82546 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537070 87 547 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537792 36 548 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537793 35549 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537794 35 550 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537795 33 551 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537796 49552 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537797 54 553 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537798 68 554 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537799 72555 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537800 69 556 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537801 82 557 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537802 72558 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537803 72 559 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537804 67 560 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537805 74561 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537806 70 562 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537809 60 563 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537810 71564 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537811 69 565 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537812 80 566 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537813 74567 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537814 54 568 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537837 70 569 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537838 76570 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537839 76 571 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537840 80 572 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537841 81573 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537842 75 574 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537843 70 575 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537844 73576 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537845 59 577 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537846 51 578 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537847 52579 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds537848 41 580 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537849 44 581 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538160 69582 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538172 24 583 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538173 23 584 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538185 68585 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538187 69 585 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538189 81 587 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538191 66588 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538192 59 589 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538193 16 590 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538194 10591 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538195 15 592 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538196  3 593 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538197 36594 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538198 49 595 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538199 47 596 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538200 57597 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538201 71 598 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538202 60 599 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538203 55600 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538204 62 601 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538205 68 602 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538228 63603 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538229 26 604 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538230 75 605 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538231 75606 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538233 52 607 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538235 26 608 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538237 28609 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538239 54 610 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538241 73 611 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538242 68612 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538243 61 613 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538245 75 614 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538253 37615 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538254 45 616 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538361 56 617 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538378 70618 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds538380 68 619 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538381 57 620 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540361 71621 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540362 73 622 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540363 78 623 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540364 89624 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540365 83 625 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540366 84 626 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540367 65627 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540368 55 628 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540369 82 629 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540370 86630 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540371 74 631 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540372 82 632 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540373 81633 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540374 87 634 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540375 78 635 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540376 69636 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540377 88 637 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540378 85 638 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540379 77639 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540380 84 640 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540381 85 641 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540382 69642 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540383 85 643 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540384 88 644 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540385 87645 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540386 86 646 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540387 77 647 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540388 86648 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540389 86 649 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540390 85 650 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540391 83651 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540392 43 652 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540393 88 653 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540394 68654 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540395 87 655 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540396 87 656 6Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540397 59657 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds540398 36 658 6 Nds Nds Nks Nks NkNes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540399 81 659 6Nds Nds Nks Nks Nk

Example 23 High Dose Tolerability of Modified OligonucleotidesComprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside(e.g cEt) Modifications Targeting Human Target-X in BALB/c Mice

BALB/c mice were treated at a high dose with ISIS antisenseoligonucleotides selected from studies described above and evaluated forchanges in the levels of various plasma chemistry markers.

Additionally, the newly designed antisense oligonucleotides were createdwith the same sequences as the antisense oligonucleotides from the studydescribed above and were also added to this screen targeting intronicrepeat regions of Target-X.

The newly designed modified antisense oligonucleotides and their motifsare described in Table 38. The internucleoside linkages throughout eachgapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d”indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate6′-(S)—CH3 bicyclic nucleoside (e.g cEt) nucleosides. Nucleosidesfollowed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides. “N”indicates modified or naturally occurring nucleobases (A, T, C, G, U, or5-methyl C).

Each gapmer listed in Table 38 is targeted to the intronic region ofhuman Target-X genomic sequence, designated herein as Target-X. “Startsite” indicates the 5′-most nucleoside to which the gapmer is targetedin the human gene sequence. “Stop site” indicates the 3′-most nucleosideto which the gapmer is targeted human gene sequence.

TABLE 38 Modified antisense oligonucleotides targeted to Target-X SEQISIS SEQ ID Sequence (5′ to 3′) No CODE NONks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537721509 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne537738 524 6Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537759539 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne537761 541 6Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537763543 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne537850 548 6Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537858556 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne537864 562 6Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537869565 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne537872 568 6Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537897571 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne540118 582 6Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540138602 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne540139 603 6Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540148612 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne540153 617 6Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540155619 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne540162 624 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540164626 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne540168 630 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540172634 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne540175 637 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540176638 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne540178 640 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540179641 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne540181 643 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540182644 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne540183 645 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540184646 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne540186 648 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540187649 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne540188 650 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540191653 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne540193 655 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540194656 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne544811 547 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544812545 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne544813 527 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544814557 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne544815 546 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544816573 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne544817 572 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544818566 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne544819 510 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544820525 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne544821 567 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544826537 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne544827 538 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544828539 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne544829 540 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544830541 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne545471 542 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545472543 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne545473 544 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545474558 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne545475 559 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545476560 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne545477 561 6Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545478562 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne545479 556 6Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537727514 6

Treatment

Male BALB/c mice were injected subcutaneously with a single dose of 200mg/kg of ISIS 422142, ISIS 457851, ISIS 473294, ISIS 473295, ISIS473327, ISIS 484714, ISIS 515334, ISIS 515338, ISIS 515354, ISIS 515366,ISIS 515380, ISIS 515381, ISIS 515382, ISIS 515384, ISIS 515386, ISIS515387, ISIS 515388, ISIS 515406, ISIS 515407, ISIS 515408, ISIS 515422,ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515533, ISIS 515534, ISIS515538, ISIS 515539, ISIS 515558, ISIS 515656, ISIS 515575, ISIS 515926,ISIS 515944, ISIS 515945, ISIS 515948, ISIS 515949, ISIS 515951, ISIS515952, ISSI 516003, ISIS 516055, ISIS 516057, ISIS 516060, ISIS 516062,ISIS 529126, ISIS 529146, ISIS 529166, ISIS 529170, ISIS 529172, ISIS529173, ISIS 529174, ISIS 529175, ISSI 529176, ISIS 529182, ISIS 529183,ISIS 529186, ISIS 529282, ISIS 529304, ISIS 529306, ISIS 529360, ISIS529450, ISIS 529459, ISIS 529460, ISIS 529461, ISIS 529547, ISIS 529550,ISIS 529551, ISIS 529553, ISIS 529557, ISIS 529562, ISIS 529563, ISIS529564, ISIS 529565, ISIS 529575, ISIS 529582, ISIS 529589, ISIS 529607,ISIS 529614, ISIS 529632, ISIS 529650, ISIS 529651, ISIS 529657, ISIS529663, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785, ISIS 529804,ISIS 529818, ISIS 529823, ISIS 529854, ISIS 534528, ISIS 534534, ISIS534594, ISIS 534660, ISIS 534663, ISIS 534664, ISIS 534676, ISIS 534677,ISIS 537679, ISIS 537683, ISIS 534693, ISIS 534701, ISIS 534716, ISIS534730, ISIS 534765, ISIS 534795, ISIS 534796, ISIS 534797, ISIS 534798,ISIS 534799, ISIS 534800, ISIS 534802, ISIS 534806, ISSI 534830, ISIS534838, ISIS 534888, ISIS 534890, ISIS 534898, ISIS 534911, ISIS 534920,ISIS 534926, ISIS 534937, ISIS 534950, ISSI 534956, ISIS 534980, ISIS534986, ISIS 535010, ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082,ISSI 535142, ISIS 537024, ISIS 537030, ISIS 537041, ISIS 537062, ISIS537064, ISIS 537066, ISIS 537721, ISIS 537727, ISIS 537738, ISIS 537759,ISIS 537761, ISIS 537763, ISIS 537792, ISIS 537800, ISIS 537806, ISIS537811, ISIS 537814, ISIS 537839, ISIS 537850, ISSI 537858, ISIS 537864,ISIS 537869, ISIS 537872, ISIS 537897, ISIS 538160, ISIS 538196, ISIS538205, ISIS 538228, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 540118,ISIS 540138, ISIS 540139, ISIS 540148, ISIS 540153, ISIS 540155, ISIS540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS 540175, ISIS 540176,ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS 540191, ISIS 540193,ISIS 540194, ISIS 544811, ISIS 544812, ISIS 544813, ISIS 544814, ISIS544815, ISIS 544816, ISIS 544817, ISIS 544818, ISIS 544819, ISIS 544820,ISIS 544821, ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS544830, ISIS 545471, ISIS 545472, ISIS 545473, ISIS 545474, ISIS 545475,ISIS 545476, ISIS 545477, ISIS 545478, and ISIS 545479. One set of maleBALB/c mice was injected with a single dose of PBS. Mice were euthanized96 hours later, and organs and plasma were harvested for furtheranalysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels oftransaminases, or which caused an increase within three times the upperlimit of normal (ULN) were deemed very tolerable. ISIS oligonucleotidesthat caused an increase in the levels of transaminases between threetimes and seven times the ULN were deemed tolerable. Based on thesecriteria, ISIS 529166, ISIS 529170, ISIS 529175, ISIS 529176, ISIS529186, ISIS 529282, ISIS 529360, ISIS 529450, ISIS 529459, ISIS 529460,ISIS 529547, ISIS 529549, ISIS 529551, ISIS 529553, ISIS 529557, ISIS529562, ISIS 529575, ISIS 529582, ISIS 529607, ISIS 529589, ISIS 529632,ISIS 529657, ISIS 529725, ISIS 529745, ISIS 529785, ISIS 529799, ISIS529804, ISIS 529818, ISIS 529823, ISIS 534950, ISIS 534980, ISIS 535010,ISIS 537030, ISIS 537041, ISIS 537062, ISIS 537064, ISIS 537066, ISIS537759, ISIS 537792, ISIS 537800, ISIS 537839, ISIS 538228, ISIS 473294,ISIS 473295, ISIS 484714, ISIS 515338, ISIS 515366, ISIS 515380, ISIS515381, ISIS 515387, ISIS 515408, ISIS 515423, ISIS 515424, ISIS 515532,ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515575, ISIS515926, ISIS 515944, ISIS 515945, ISIS 515951, ISIS 515952, ISIS 529126,ISIS 529765, ISIS 534528, ISIS 534534, ISIS 534594, ISIS 534663, ISIS534676, ISIS 534677, ISIS 534679, ISIS 534683, ISIS 534693, ISIS 534701,ISIS 534716, ISIS 534730, ISIS 534806, ISIS 534830, ISIS 534838, ISIS534890, ISIS 534898, ISIS 534911, ISIS 534937, ISIS 534956, ISIS 534986,ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082, ISIS 535142, ISIS538160, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 534795, ISIS 534796,ISIS 534797, ISIS 540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS540175, ISIS 540176, ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182,ISIS 540183, ISIS 540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS540191, ISIS 540193, ISIS 540194, ISIS 544813, ISIS 544814, ISIS 544816,ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS 545473, andISIS 545474 were considered very tolerable in terms of liver function.Based on these criteria, ISIS 529173, ISIS 529854, ISIS 529614, ISIS515386, ISIS 515388, ISIS 515949, ISIS 544817, and ISIS 545479 wereconsidered tolerable in terms of liver function.

Example 24 Tolerability of Antisense Oligonucleotides Targeting HumanTarget-X in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety andefficacy evaluations. The rats were treated with ISIS antisenseoligonucleotides from the studies described in the Examples above andevaluated for changes in the levels of various plasma chemistry markers.

Treatment

Six-eight week old male Sprague-Dawley rats were maintained on a 12-hourlight/dark cycle and fed ad libitum with Teklad normal rat chow. Groupsof four Sprague-Dawley rats each were injected subcutaneously twice aweek for 6 weeks with 25 mg/kg of ISIS 473286, ISIS 473547, ISIS 473567,ISIS 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS515641, ISIS 515655, ISIS 515657, ISIS 516046, ISIS 516048, ISIS 516051,ISIS 516052, and ISIS 516062. A group of four Sprague-Dawley rats wasinjected subcutaneously twice a week for 6 weeks with PBS. Forty eighthours after the last dose, rats were euthanized and organs and plasmawere harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasmalevels of ALT (alanine transaminase) and AST (aspartate transaminase)were measured. Plasma levels of Bilirubin and BUN were also measuredusing the same clinical chemistry analyzer.

ISIS oligonucleotides that did not cause any increase in the levels oftransaminases, or which caused an increase within three times the upperlimit of normal (ULN) were deemed very tolerable. ISIS oligonucleotidesthat caused an increase in the levels of transaminases between threetimes and seven times the ULN were deemed tolerable. Based on thesecriteria, ISIS 473286, ISIS 473547, ISSI 473589, ISIS 473630, ISIS484559, ISIS 515636, ISIS 515640, ISIS 515655, ISIS 516046, and ISIS516051 were considered very tolerable in terms of liver function. Basedon these criteria, ISIS 473567, ISIS 515641, ISIS 515657, ISIS 516048,and ISIS 516051 were considered tolerable in terms of liver function.

Example 25 Tolerability of Chimeric Antisense OligonucleotidesComprising 2′-O-Methoxyethyl (2′-MOE) Modifications Targeting HumanTarget-X in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotidesfrom the studies described in the Examples above and evaluated forchanges in the levels of various plasma chemistry markers.

Treatment

Six-eight week old male Sprague-Dawley rats were maintained on a 12-hourlight/dark cycle and fed ad libitum with Purina normal rat chow. Groupsof four Sprague-Dawley rats each were injected subcutaneously twice aweek for 6 weeks with 50 mg/kg of ISIS 407936, ISIS 416507, ISIS 416508,ISIS 490208, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS490803, ISIS 491122, ISIS 513419, ISIS 513446, ISIS 513454, ISIS 513455,ISIS 513456, ISIS 513504, ISIS 513507, and ISIS 513508. A group of fourSprague-Dawley rats was injected subcutaneously twice a week for 6 weekswith PBS. Forty eight hours after the last dose, rats were euthanizedand organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasmalevels of Bilirubin and BUN were also measured using the same clinicalchemistry analyzer.

ISIS oligonucleotides that did not cause any increase in the levels oftransaminases, or which caused an increase within three times the upperlimit of normal (ULN) were deemed very tolerable. ISIS oligonucleotidesthat caused an increase in the levels of transaminases between threetimes and seven times the ULN were deemed tolerable. Based on thesecriteria, ISIS 416507, ISIS 490208, ISIS 490368, ISIS 490396, ISIS490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456,ISIS 513504, and ISIS 513508 were considered very tolerable in terms ofliver function. Based on these criteria, ISIS 407936, ISIS 416508, ISIS490279, and ISIS 513507 were considered tolerable in terms of liverfunction.

Example 26 Tolerability of Chimeric Antisense OligonucleotidesComprising 2′-O-Methoxyethyl (2′-MOE) Modifications Targeting HumanTarget-X in CD-1 Mice

CD-1 mice are a multipurpose mice model, frequently utilized for safetyand efficacy testing. The mice were treated with ISIS antisenseoligonucleotides selected from studies described above and evaluated forchanges in the levels of various plasma chemistry markers.

Treatment

Groups of 3 male CD-1 mice each were injected subcutaneously twice aweek for 6 weeks with 50 mg/kg of ISIS 473244, ISIS 473295, ISIS 484714,ISIS 515386, ISIS 515424, ISIS 515534, ISIS 515558, ISIS 515926, ISIS515949, ISIS 515951, ISIS 515952, ISIS 529126, ISIS 529166, ISIS 529173,ISIS 529186, ISIS 529360, ISIS 529461, ISIS 529553, ISIS 529564, ISIS529582, ISIS 529614, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785,ISIS 529799, ISIS 529818, ISIS 529823, ISIS 534528, ISIS 534594, andISIS 534664. One group of male CD-1 mice was injected subcutaneouslytwice a week for 6 weeks with PBS. Mice were euthanized 48 hours afterthe last dose, and organs and plasma were harvested for furtheranalysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels oftransaminases, or which caused an increase within three times the upperlimit of normal (ULN) were deemed very tolerable. ISIS oligonucleotidesthat caused an increase in the levels of transaminases between threetimes and seven times the ULN were deemed tolerable. Based on thesecriteria, ISIS 473295, ISIS 473714, ISIS 515558, ISIS 515926, 515951,ISIS 515952, ISIS 529126, ISIS 529166, 529564, ISIS 529582, ISIS 529614,ISIS 529725, ISIS 529765, ISIS 529799, ISIS 529823, and ISIS 534594 wereconsidered very tolerable in terms of liver function. Based on thesecriteria, ISIS 515424, ISIS 515534, ISIS 515926, ISIS 529785, and ISIS534664 were considered tolerable in terms of liver function.

Example 27 Tolerability of Chimeric Antisense OligonucleotidesComprising 2′-O-Methoxyethyl (2′-MOE) Modifications Targeting HumanTarget-X in CD-1 Mice

CD-1 mice were treated with ISIS antisense oligonucleotides selectedfrom studies described above and evaluated for changes in the levels ofvarious plasma chemistry markers.

Treatment

Groups of 3 male CD-1 mice each were injected subcutaneously twice aweek for 6 weeks with 100 mg/kg of ISIS 490208, ISIS 490279, ISIS490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513419,ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, ISIS513507, and ISIS 513508. Groups of 3 male CD-1 mice each were injectedsubcutaneously twice a week for 6 weeks with 100 mg/kg of ISIS 407936,ISIS 416507, and ISIS 416508, which are gapmers described in a previouspublication. One group of male CD-1 mice was injected subcutaneouslytwice a week for 6 weeks with PBS. Mice were euthanized 48 hours afterthe last dose, and organs and plasma were harvested for furtheranalysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, and BUN weremeasured using an automated clinical chemistry analyzer (Hitachi OlympusAU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels oftransaminases, or which caused an increase within three times the upperlimit of normal (ULN) were deemed very tolerable. ISIS oligonucleotidesthat caused an increase in the levels of transaminases between threetimes and seven times the ULN were deemed tolerable. Based on thesecriteria, ISIS 407936, ISIS 416507, ISIS 490279, ISIS 490368, ISIS490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513456,and ISIS 513504 were considered very tolerable in terms of liverfunction. Based on these criteria, ISIS 490208, ISIS 513455, ISIS513507, and ISIS 513508 were considered tolerable in terms of liverfunction.

Example 28 Efficacy of Modified Oligonucleotides Comprising2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt)Modifications Targeting Human Target-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotidesselected from studies described above and evaluated for efficacy.

Treatment

Groups of 2-3 male and female transgenic mice were injectedsubcutaneously twice a week for 3 weeks with 5 mg/kg/week of ISIS473244, ISIS 473295, ISIS 484714, ISIS 515926, ISIS 515951, ISIS 515952,ISIS 516062, ISIS 529126, ISIS 529553, ISIS 529745, ISIS 529799, ISIS534664, ISIS 534826, ISIS 540168, ISIS 540175, ISIS 544826, ISIS 544827,ISIS 544828, and ISIS 544829. One group of mice was injectedsubcutaneously twice a week for 3 weeks with PBS. Mice were euthanized48 hours after the last dose, and organs and plasma were harvested forfurther analysis.

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISAkit (purchased from Hyphen Bio-Med). Results are presented as percentinhibition of Target-X, relative to control. As shown in Table 39,several antisense oligonucleotides achieved reduction of human Target-Xover the PBS control. ‘n.d.’ indicates that the value for thatparticular oligonucleotide was not measured.

TABLE 39 Percent inhibition of Target-X plasma protein levels intransgenic mice ISIS No % inhibition 473244 2 473295 13 484714 19 51592611 515951 13 515952 0 516062 62 529126 0 529553 0 529745 22 529799 26534664 32 534826 n.d. 540168 94 540175 98 544813 0 544826 23 544827 60544828 33 544829 53

Example 29 Efficacy of Modified Oligonucleotides Comprising2′-Methoxyethyl (2′-MOE) and 6′-(S)—CH₃ Bicyclic Nucleoside (e.g cEt)Modifications Targeting Human Target-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotidesselected from studies described above and evaluated for efficacy.

Treatment

Groups of 2-3 male and female transgenic mice were injectedsubcutaneously twice a week for 3 weeks with 1 mg/kg/week of ISIS407936, ISIS 490197, ISIS 490275, ISIS 490278, ISIS 490279, ISIS 490323,ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS513447, ISIS 513504, ISIS 516062, ISIS 529166, ISIS 529173, ISIS 529360,ISIS 529725, ISIS 534557, ISIS 534594, ISIS 534664, ISIS 534688, ISIS534689, ISIS 534915, ISIS 534916, ISIS 534917, and ISIS 534980. Onegroup of mice was injected subcutaneously twice a week for 3 weeks withPBS. Mice were euthanized 48 hours after the last dose, and organs andplasma were harvested for further analysis.

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISAkit (purchased from Hyphen Bio-Med). Results are presented as percentinhibition of Target-X, relative to control. As shown in Table 40,several antisense oligonucleotides achieved reduction of human Target-Xover the PBS control.

TABLE 40 Percent inhibition of Target-X plasm protein levels intransgenic mice ISIS No % inhibition 407936 28 490197 50 490275 21490278 20 490279 59 490323 54 490368 22 490396 31 490803 30 491122 51513446 29 513447 44 513504 45 516062 75 529166 37 529173 64 529360 43529725 53 534557 76 534594 40 534664 14 534687 12 534688 48 534689 25534915 40 534916 45 534917 66 534980 62

Example 30 Tolerability of Antisense Oligonucleotides Targeting HumanTarget-X in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotidesfrom the studies described in the Examples above and evaluated forchanges in the levels of various plasma chemistry markers.

Treatment

Six-eight week old male Sprague-Dawley rats were maintained on a 12-hourlight/dark cycle and fed ad libitum with Teklad normal rat chow. Groupsof four Sprague-Dawley rats each were injected subcutaneously twice aweek for 4 weeks with ISIS 515380, ISIS 515381, ISIS 515387, ISIS529175, ISIS 529176, ISIS 529575, ISIS 529804, and ISIS 537064. Doses 1,5, 6, 7, and 8 were 25 mg/kg; dose 2 was 75 mg/kg; doses 3 and 4 were 50mg/kg. One group of four Sprague-Dawley rats was injected subcutaneouslytwice a week for 4 weeks with PBS. Forty eight hours after the lastdose, rats were euthanized and organs and plasma were harvested forfurther analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasmalevels of ALT (alanine transaminase) and AST (aspartate transaminase)were measured. Plasma levels of Bilirubin and BUN were also measuredusing the same clinical chemistry analyzer.

ISIS oligonucleotides that did not cause any increase in the levels oftransaminases, or which caused increase in the levels within three timesthe upper limit of normal levels of transaminases were deemed verytolerable. ISIS oligonucleotides that caused increase in the levels oftransaminases between three times and seven times the upper limit ofnormal levels were deemed tolerable. Based on these criteria, ISIS515380, ISIS 515387, ISIS 529175, ISIS 529176, ISIS 529804, and ISIS537064 were considered very tolerable in terms of liver function. Basedon these criteria, ISIS 515381 was considered tolerable in terms ofliver function.

Example 31 Efficacy of Antisense Oligonucleotides Targeting HumanTarget-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotidesselected from studies described above and evaluated for efficacy.

Treatment

Two groups of 3 male and female transgenic mice were injectedsubcutaneously twice a week for 2 weeks with 0.5 mg/kg/week or 1.5mg/kg/week of ISIS 407935 and ISIS 513455. Another group of mice wassubcutaneously twice a week for 2 weeks with 0.6 mg/kg/week or 2.0mg/kg/week of ISIS 473286. Another 16 groups of mice were subcutaneouslytwice a week for 2 weeks with 0.1 mg/kg/week or 0.3 mg/kg/week of ISIS473589, ISIS 515380, ISIS 515423, ISIS 529804, ISIS 534676, ISIS 534796,ISIS 540162, ISIS 540164, ISIS 540175, ISIS 540179, ISIS 540181, ISIS540182, ISIS 540186, ISIS 540191, ISIS 540193, ISIS 544827, or ISIS545474. Another 3 groups of mice were injected subcutaneously twice aweek for 2 weeks with 0.3 mg/kg/week of ISIS 516062, ISIS 534528 or ISIS534693. One group of mice was injected subcutaneously twice a week for 2weeks with PBS. Mice were euthanized 48 hours after the last dose, andorgans and plasma were harvested for further analysis.

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISAkit (purchased from Hyphen Bio-Med). Results are presented as percentinhibition of Target-X, relative to control. As shown in Table 41,several antisense oligonucleotides achieved reduction of human Target-Xover the PBS control.

TABLE 41 Percent inhibition of Target-X plasma protein levels intransgenic mice Dose % ISIS No (mg/kg/wk) inhibition 407935 1.5 65 0.531 513455 1.5 64 0.5 52 473286 2 67 0.6 11 473589 0.3 42 0.1 12 5153800.3 64 0.1 32 515423 0.3 72 0.1 37 529804 0.3 36 0.1 24 534676 0.3 310.1 18 534796 0.3 54 0.1 43 540162 0.3 84 0.1 42 540164 0.3 25 0.1 17540175 0.3 90 0.1 55 540179 0.3 29 0.1 24 540181 0.3 53 0.1 0 540182 0.378 0.1 21 540186 0.3 72 0.1 46 540191 0.3 62 0.1 35 540193 0.3 74 0.1 46544827 0.3 28 0.1 19 545474 0.3 59 0.1 0 516062 0.3 33 534528 0.3 41534693 0.3 34

Example 32 Tolerability of Antisense Oligonucleotides Targeting HumanTarget-X in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotidesfrom the studies described in the Examples above and evaluated forchanges in the levels of various plasma chemistry markers.

Treatment

Five-six week old male Sprague-Dawley rats were maintained on a 12-hourlight/dark cycle and fed ad libitum with Teklad normal rat chow. Groupsof four Sprague-Dawley rats each were injected subcutaneously twice aweek for 4 weeks with 50 mg/kg of ISIS 515423, ISIS 515424, ISIS 515640,ISIS 534676, ISIS 534796, ISIS 534797, ISIS 540162, ISIS 540164, ISIS540172, ISIS 540175, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183,ISIS 540186, ISIS 540191, and ISIS 545474. A group of fourSprague-Dawley rats was injected subcutaneously twice a week for 4 weekswith PBS. Forty eight hours after the last dose, rats were euthanizedand organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasmalevels of ALT (alanine transaminase) and AST (aspartate transaminase)were measured. Plasma levels of Bilirubin and BUN were also measuredusing the same clinical chemistry analyzer.

ISIS oligonucleotides that did not cause any increase in the levels oftransaminases, or which caused an increase within three times the upperlimit of normal (ULN) were deemed very tolerable. ISIS oligonucleotidesthat caused an increase in the levels of transaminases between threetimes and seven times the ULN were deemed tolerable. Based on thesecriteria, ISIS 540164, ISIS 540172, and ISIS 540175 were considered verytolerable in terms of liver function. Based on these criteria, ISIS534676, ISIS 534796, ISIS 534797, ISIS 540162, and ISIS 540179 wereconsidered tolerable in terms of liver function.

Example 33 Dose-Dependent Antisense Inhibition of Human Target-X inHep3B Cells

Antisense oligonucleotides selected from the studies described abovewere tested at various doses in Hep3B cells. Cells were plated at adensity of 20,000 cells per well and transfected using electroporationwith 0.05 μM, 0.15 μM, 0.44 μM, 1.33 μM, and 4.00 μM concentrations ofantisense oligonucleotide, as specified in Table 42. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andTarget-X mRNA levels were measured by quantitative real-time PCR. HumanTarget-X primer probe set RTS2927 was used to measure mRNA levels.Target-X mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN®. Results are presented as percent inhibition ofTarget-X, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 42. As illustrated in Table 42, Target-X mRNAlevels were reduced in a dose-dependent manner in several of theantisense oligonucleotide treated cells.

TABLE 42 Dose-dependent antisense inhibition of human Target-X in Hep3Bcells using electroporation 0.05 0.15 0.44 1.33 4.00 IC₅₀ ISIS No μM μMμM μM μM (μM) 473286 0 1 13 12 15 >4.0 457851 23 32 57 80 93 0.3 4732863 20 43 71 88 0.5 473286 15 26 24 28 36 >4.0 473286 6 3 10 26 29 >4.0473327 14 28 35 67 90 0.5 473589 29 53 76 89 95 0.1 515380 44 72 85 9395 <0.05 515423 43 64 87 95 98 <0.05 515424 38 55 85 92 97 0.1 515636 2133 74 82 93 0.2 516046 29 23 29 48 78 0.9 516048 35 24 41 67 87 0.4516052 18 6 48 63 80 0.6 516062 24 14 21 47 68 1.6 529166 16 47 75 87 940.2 529173 14 49 77 91 96 0.2 529175 30 69 88 93 96 0.1 529176 34 63 8593 96 0.1 529360 35 53 74 91 93 0.1 529725 53 69 85 92 95 <0.05 52980437 41 71 90 94 0.1 534528 50 68 78 93 97 <0.05 534557 48 78 90 94 95<0.05 534594 39 47 76 87 94 0.1 534676 29 20 40 64 87 0.5 534687 41 3756 80 93 0.2 534688 16 56 88 94 96 0.1 534689 21 59 82 94 95 0.1 53469318 58 81 93 95 0.1 534795 19 43 68 90 94 0.2 534796 25 59 80 93 96 0.1534890 31 55 77 90 96 0.1 534898 22 61 80 94 97 0.1 534915 19 26 51 7794 0.3 534916 20 36 66 86 93 0.2 534917 34 53 82 89 94 0.1 540162 40 6484 90 92 <0.05 540164 34 60 83 91 92 0.1 540168 51 79 90 92 94 <0.05540172 40 66 80 88 92 <0.05 540175 30 61 80 88 91 0.1 540176 7 17 50 7585 0.5 540179 11 22 25 16 19 >4.0 540181 19 46 72 86 91 0.2 540182 16 6683 86 92 0.1 540183 39 74 87 92 93 <0.05 540186 31 69 85 91 94 0.1540191 38 54 80 88 91 0.1 540193 57 67 84 94 97 <0.05 540194 30 45 62 7791 0.2 544827 37 42 67 82 96 0.1 544829 26 41 42 71 93 0.3 545473 28 2749 80 97 0.3 545474 23 27 55 84 96 0.3

Example 34 Tolerability of Antisense Oligonucleotides Targeting HumanTarget-X in CD-1 Mice

CD-1 mice were treated with ISIS antisense oligonucleotides selectedfrom studies described above and evaluated for changes in the levels ofvarious plasma chemistry markers.

Treatment

Two groups of 4 male 6-8 week old CD-1 mice each were injectedsubcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 andISIS 490279. Another seven groups of 4 male 6-8 week old CD-1 mice eachwere injected subcutaneously twice a week for 6 weeks with 25 mg/kg ofISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS540182, and ISIS 540191. One group of male CD-1 mice was injectedsubcutaneously twice a week for 6 weeks with PBS. Mice were euthanized48 hours after the last dose, and organs and plasma were harvested forfurther analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.). The results are presented in Table 43.Treatment with the newly designed antisense oligonucleotides were moretolerable compared to treatment with ISIS 407935 (disclosed in anearlier publication), which caused elevation of ALT levels greater thanseven times the upper limit of normal (ULN).

TABLE 43 Effect of antisense oligonucleotide treatment on liver functionin CD-1 mice Dose AST BUN Bilirubin Motif (mg/kg/wk) ALT(IU/L) (IU/L)(mg/dL) (mg/dL) PBS — — 37 47 28 0.2 407935 e5-d(10)-e5 100 373 217 240.2 490279 kdkdk-d(9)-ee 100 96 82 24 0.2 473589 e5-d(10)-e5 50 93 11622 0.2 529804 k-d(10)-kekee 50 54 74 27 0.2 534796 ekk-d(10)-kke 50 6063 27 0.2 540162 eek-d(10)-kke 50 43 55 29 0.2 540175 eek-d(10)-kke 50113 78 24 0.3 540182 eek-d(10)-kke 50 147 95 26 0.1 540191 eek-d(10)-kke50 79 88 28 0.2 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Body and Organ Weights

Body weights, as well as liver, heart, lungs, spleen and kidney weightswere measured at the end of the study, and are presented in Table 44.Several of the ISIS oligonucleotides did not cause any changes in organweights outside the expected range and were therefore deemed tolerablein terms of organ weights.

TABLE 44 Body and organ weights (grams) of CD-1 mice Dose (mg/ Body Liv-Kid- Motif kg/wk) weight er Spleen ney PBS — — 42 2.2 0.12 0.64 407935e5-d(10)-e5 100 40 2.6 0.20 0.62 490279 kdkdk-d(9)-ee 100 42 2.8 0.170.61 473589 e5-d(10)-e5 50 41 2.5 0.16 0.67 529804 k-d(10)-kekee 50 402.3 0.14 0.62 534796 ekk-d(10)-kke 50 37 2.6 0.15 0.51 540162eek-d(10)-kke 50 42 2.4 0.15 0.60 540175 eek-d(10)-kke 50 39 2.2 0.110.62 540182 eek-d(10)-kke 50 41 2.6 0.16 0.61 540191 eek-d(10)-kke 50 402.4 0.13 0.60 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 35 Tolerability of Antisense Oligonucleotides Targeting HumanTarget-X in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotidesselected from studies described above and evaluated for changes in thelevels of various plasma chemistry markers.

Treatment

Two groups of 4 male 7-8 week old Sprague-Dawley rats each were injectedsubcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 andISIS 490279. Another seven groups of 4 male 6-8 week old Sprague-Dawleyrats each were injected subcutaneously twice a week for 6 weeks with 25mg/kg of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS540175, ISIS 540182, and ISIS 540191. One group of male Sprague-Dawleyrats was injected subcutaneously twice a week for 6 weeks with PBS. Therats were euthanized 48 hours after the last dose, and organs and plasmawere harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.). The results are presented in Table 45.Treatment with the all antisense oligonucleotides was tolerable in termsof plasma chemistry markers in this model.

TABLE 45 Effect of antisense oligonucleotide treatment on liver functionin Sprague-Dawley rats Dose AST BUN Bilirubin Motif (mg/kg/wk) ALT(IU/L)(IU/L) (mg/dL) (mg/dL) PBS — — 71 83 19 0.2 407935 e5-d(10)-e5 100 74 9622 0.2 490279 kdkdk-d(9)-ee 100 96 181 22 0.4 473589 e5-d(10)-e5 50 5773 21 0.2 529804 k-d(10)-kekee 50 54 78 21 0.2 534796 ekk-d(10)-kke 5068 98 22 0.2 540162 eek-d(10)-kke 50 96 82 21 0.1 540175 eek-d(10)-kke50 55 73 18 0.2 540182 eek-d(10)-kke 50 45 87 21 0.2 540191eek-d(10)-kke 50 77 104 21 0.2 e = 2′-MOE, k = cEt, d =2′-deoxynucleoside

Body and Organ Weights

Body weights, as well as liver, heart, lungs, spleen and kidney weightswere measured at the end of the study, and are presented in Table 46.Treatment with all the antisense oligonucleotides was tolerable in termsof body and organ weights in this model.

TABLE 46 Body and organ weights (grams) of Sprague-Dawley rats Dose (mg/Body Liv- Kid- Motif kg/wk) weight er Spleen ney PBS — — 443 16 0.8 3.5ISIS 407935 e5-d(10)-e5 100 337 14 1.8 3.2 ISIS 490279 kdkdk-d(9)-ee 100365 18 2.2 2.9 ISIS 473589 e5-d(10)-e5 50 432 18 1.3 3.3 ISIS 529804k-d(10)-kekee 50 429 18 2.2 3.4 ISIS 534796 ekk-d(10)-kke 50 434 15 1.43.3 ISIS 540162 eek-d(10)-kke 50 446 18 1.1 3.3 ISIS 540175eek-d(10)-kke 50 467 16 1.0 3.5 ISIS 540182 eek-d(10)-kke 50 447 22 2.54.5 ISIS 540191 eek-d(10)-kke 50 471 21 1.4 3.9 e = 2′-MOE, k = cEt, d =2′-deoxynucleoside

Example 36 Dose-Dependent Antisense Inhibition of Human Target-X inCynomolgos Monkey Primary Hepatocytes

Antisense oligonucleotides selected from the studies described abovewere tested at various doses in cynomolgus monkey primary hepatocytes.Cells were plated at a density of 35,000 cells per well and transfectedusing electroporation with 0.009 μM, 0.03 μM, 0.08 μM, 0.25 μM, 0.74 μM,2.22 μM, 6.67 μM, and 20.00 μM concentrations of antisenseoligonucleotide, as specified in Table 47. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells and Target-XmRNA levels were measured by quantitative real-time PCR. Target-X primerprobe set RTS2927 was used to measure mRNA levels. Target-X mRNA levelswere adjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of Target-X, relative tountreated control cells. As illustrated in Table 47, Target-X mRNAlevels were reduced in a dose-dependent manner with some of theantisense oligonucleotides that are cross-reactive with the rhesusmonkey genomic sequence.

TABLE 47 Dose-dependent antisense inhibition of Target-X in cynomolgousmonkey primary hepatocytes using electroporation 0.009 0.03 0.08 0.250.74 2.22 6.67 20.00 ISIS No μM μM μM μM μM μM μM μM 407935 10 18 15 2956 73 82 88 490279 19 12 13 0 6 18 27 22 473589 5 10 19 42 64 76 88 92529804 10 3 23 25 57 80 86 91 534796 0 28 23 49 71 81 87 90 540162 9 149 6 13 13 11 31 540175 0 4 12 9 10 16 12 22 540182 0 7 0 6 36 12 10 0540191 6 7 0 0 0 0 21 42

Example 37 Dose-Dependent Antisense Inhibition of Human Target-X inHep3B Cells

Antisense oligonucleotides from the study described above were alsotested at various doses in Hep3B cells. Cells were plated at a densityof 20,000 cells per well and transfected using electroporation with0.009 μM, 0.03 μM, 0.08 μM, 0.25 μM, 0.74 μM, 2.22 μM, 6.67 μM, and20.00 μM concentrations of antisense oligonucleotide, as specified inTable 48. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and Target-X mRNA levels were measured byquantitative real-time PCR. Target-X primer probe set RTS2927 was usedto measure mRNA levels. Target-X mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of Target-X, relative to untreated control cells. Asillustrated in Table 48, Target-X mRNA levels were reduced in adose-dependent manner with several of the antisense oligonucleotides.

TABLE 48 Dose-dependent antisense inhibition of Target-X in Hep3B cellsusing electroporation 0.009 0.03 0.08 0.25 0.74 2.22 6.67 20.00 IC₅₀ISIS No μM μM μM μM μM μM μM μM (μM) 407935 3 9 11 35 64 83 87 93 4.5473244 20 33 50 69 77 89 7 14 0.9 473589 0 14 23 44 74 88 90 94 2.7490279 0 5 7 15 25 61 76 78 11.6 515533 0 12 21 36 63 78 88 94 3.6515952 0 12 27 57 76 89 93 94 2.2 516066 6 0 12 26 52 70 81 86 6.0529459 0 4 24 40 61 78 88 94 3.5 529553 9 7 17 40 58 74 87 93 4.6 5298040 3 34 64 83 89 93 95 2.0 534796 8 18 43 67 82 89 95 96 1.4 537806 6 115 20 37 69 79 86 7.1 540162 18 33 63 75 87 91 91 92 0.7 540175 10 25 5576 86 89 89 93 1.0 540182 13 36 61 75 84 88 90 93 0.7 540191 3 12 28 6179 80 88 94 2.2

Example 38 Efficacy of Antisense Oligonucleotides Targeting HumanTarget-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotidesselected from studies described above and evaluated for efficacy.

Treatment

Eight groups of 3 transgenic mice each were injected subcutaneouslytwice a week for 3 weeks with 20 mg/kg/week, 10 mg/kg/week, 5mg/kg/week, or 2.5 mg/kg/week of ISIS 407935 or ISIS 490279. Another 24groups of 3 transgenic mice each were subcutaneously twice a week for 3weeks with 5 mg/kg/week, 2.5 mg/kg/week, 1.25 mg/kg/week, or 0.625mg/kg/week of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS540175, or ISIS 540191. One group of mice was injected subcutaneouslytwice a week for 3 weeks with PBS. Mice were euthanized 48 hours afterthe last dose, and organs and plasma were harvested for furtheranalysis.

RNA Analysis

RNA was extracted from plasma for real-time PCR analysis of Target-X,using primer probe set RTS2927. The mRNA levels were normalized usingRIBOGREEN®. As shown in Table 49, several antisense oligonucleotidesachieved reduction of human Target-X over the PBS control. Results arepresented as percent inhibition of Target-X, relative to control.Treatment with newly designed 2′-MOE gapmer, ISIS 490279, caused greaterreduction in human Target-X mRNA levels than treatment with ISIS 407935,the 2′-MOE gapmer from the earlier publication. Treatment with severalof the newly designed oligonucleotides also caused greater reduction inhuman Target-X mRNA levels than treatment with ISIS 407935.

TABLE 49 Percent inhibition of Target-X mRNA in transgenic mice Dose %ISIS No Motif (mg/kg/wk) inhibition 407935 e5-d(10)-e5 20.0 85 10.0 575.0 45 2.5 28 490279 kdkdk-d(9)-ee 20.0 88 10.0 70 5.0 51 2.5 33 473589e5-d(10)-e5 5.00 80 2.50 62 1.25 44 0.625 25 529804 k-d(10)-kekee 5.0055 2.50 41 1.25 0 0.625 1 534796 ekk-d(10)-kke 5.00 56 2.50 41 1.25 50.625 0 540162 eek-d(10)-kke 5.00 97 2.50 92 1.25 69 0.625 78 540175eek-d(10)-kke 5.00 95 2.50 85 1.25 65 0.625 55 540182 eek-d(10)-kke 5.0097 2.50 83 1.25 54 0.625 10 540191 eek-d(10)-kke 5.00 91 2.50 74 1.25 580.625 34 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISAkit (purchased from Hyphen Bio-Med). As shown in Table 50, severalantisense oligonucleotides achieved reduction of human Target-X over thePBS control. Results are presented as percent inhibition of Target-X,relative to control.

TABLE 50 Percent inhibition of Target-X plasm protein levels intransgenic mice Dose % ISIS No Motif (mg/kg/wk) inhibition 407935e5-d(10)-e5 20 65 10 47 5 0 2.5 3 490279 kdkdk-d(9)-ee 20 91 10 75 5 312.5 23 473589 e5-d(10)-e5 5 78 2.5 40 1.25 6 0.625 0 529804k-d(10)-kekee 5 50 2.5 36 1.25 0 0.625 8 534796 ekk-d(10)-kke 5 45 2.526 1.25 0 0.625 8 540162 eek-d(10)-kke 5 98 2.5 96 1.25 78 0.625 74540175 eek-d(10)-kke 5 93 2.5 83 1.25 49 0.625 24 540182 eek-d(10)-kke 597 2.5 71 1.25 50 0.625 0 540191 eek-d(10)-kke 5 97 2.5 74 1.25 46 0.62525 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 39 Effect of ISIS Antisense Oligonucleotides Targeting HumanTarget-X in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotidesselected from studies described above, including ISIS 407935, ISIS490279, ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175,ISIS 540182, and ISIS 540191. Antisense oligonucleotide efficacy wasevaluated. ISIS 407935, from the earlier publication, was included inthe study for comparison.

Treatment

Prior to the study, the monkeys were kept in quarantine for at least a30-day period, during which the animals were observed daily for generalhealth. Standard panels of serum chemistry and hematology, examinationof fecal samples for ova and parasites, and a tuberculosis test wereconducted immediately after the animals' arrival to the quarantine area.The monkeys were 2-4 years old at the start of treatment and weighedbetween 2 and 4 kg. Ten groups of four randomly assigned male cynomolgusmonkeys each were injected subcutaneously with ISIS oligonucleotide orPBS using a stainless steel dosing needle and syringe of appropriatesize into one of 4 sites on the back of the monkeys; each site used inclock-wise rotation per dose administered. Nine groups of monkeys weredosed four times a week for the first week (days 1, 3, 5, and 7) asloading doses, and subsequently once a week for weeks 2-12, with 35mg/kg of ISIS 407935, ISIS 490279, ISIS 473589, ISIS 529804, ISIS534796, ISIS 540162, ISIS 540175, ISIS 540182, or ISIS 540191. A controlgroup of cynomolgus monkeys was injected with PBS subcutaneously thricefour times a week for the first week (days 1, 3, 5, and 7), andsubsequently once a week for weeks 2-12. The protocols described in theExample were approved by the Institutional Animal Care and Use Committee(IACUC).

Hepatic Target Reduction RNA Analysis

On day 86, RNA was extracted from liver tissue for real-time PCRanalysis of Target-X using primer probe set RTS2927. Results arepresented as percent inhibition of Target-X mRNA, relative to PBScontrol, normalized to RIBOGREEN® or to the house keeping gene, GAPDH.As shown in Table 52, treatment with ISIS antisense oligonucleotidesresulted in reduction of Target-X mRNA in comparison to the PBS control.

TABLE 52 Percent Inhibition of cynomolgous monkey Target-X mRNA in thecynomolgus monkey liver relative to the PBS control ISIS No MotifRTS2927/Ribogreen RTS2927/GAPDH 407935 e5-d(10)-e5 90 90 490279kdkdk-d(9)-ee 72 66 473589 e5-d(10)-e5 96 96 529804 k-d(10)-kekee 90 87534796 ekk-d(10)-kke 80 78 540162 eek-d(10)-kke 66 58 540175eek-d(10)-kke 68 66 540182 eek-d(10)-kke 0 0 540191 eek-d(10)-kke 34 14e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Protein Levels and Activity Analysis

Plasma Target-X levels were measured prior to dosing, and on day 3, day5, day 7, day 16, day 30, day 44, day 65, and day 86 of treatment.Target-X activity was measured using Target-X deficient plasma.Approximately 1.5 mL of blood was collected from all available studyanimals into tubes containing 3.2% sodium citrate. The samples wereplaced on ice immediately after collection. Collected blood samples wereprocessed to platelet poor plasma and the tubes were centrifuged at3,000 rpm for 10 min at 4° C. to obtain plasma.

Protein levels of Target-X were measured by a Target-X elisa kit(purchased from Hyphen BioMed). The results are presented in Table 53.

TABLE 53 Plasma Target-X protein levels (% reduction compared to thebaseline) in the cynomolgus monkey plasma Day Day Day Day Day Day DayDay ISIS No 3 5 7 16 30 44 65 86 407935 21 62 69 82 84 85 84 90 490279 029 35 30 38 45 51 58 473589 12 67 85 97 98 98 98 98 529804 19 65 76 8788 89 90 90 534796 1 46 54 64 64 67 66 70 540162 0 24 26 37 45 49 49 50540175 0 28 36 38 47 52 55 55 540182 0 17 8 0 0 0 5 0 540191 0 12 4 0 04 9 10

Example 40 Single Nucleotide Polymorphisms (SNPs) in the Huntingtin(HTT) Gene Sequence

SNP positions (identified by Hayden et al, WO/2009/135322) associatedwith the HTT gene were mapped to the HTT genomic sequence, designatedherein as SEQ ID NO: 1 (NT_(—)006081.18 truncated from nucleotides1566000 to 1768000). Table 56 provides SNP positions associated with theHTT gene. Table 56 provides a reference SNP ID number from the EntrezSNP database at the National Center for Biotechnology Information (NCBI,http://www.ncbi.nlm.nih.gov/sites/entrez?db=snp), incorporated herein byreference. Table 56 furnishes further details on each SNP. The‘Reference SNP ID number’ or ‘RS number’ is the number designated toeach SNP from the Entrez SNP database at NCBI, incorporated herein byreference. ‘SNP position’ refers to the nucleotide position of the SNPon SEQ ID NO: 1. ‘Polymorphism’ indicates the nucleotide variants atthat SNP position. ‘Major allele’ indicates the nucleotide associatedwith the major allele, or the nucleotide present in a statisticallysignificant proportion of individuals in the human population. ‘Minorallele’ indicates the nucleotide associated with the minor allele, orthe nucleotide present in a relatively small proportion of individualsin the human population.

TABLE 56 Single Nuclear Polymorphisms (SNPs) and their positions on SEQID NO: 1 SNP Major Minor RS No. position Polymorphism allele allelers2857936 1963 C/T C T rs12506200 3707 A/G G A rs762855 14449 A/G G Ars3856973 19826 G/A G A rs2285086 28912 G/A A G rs7659144 37974 C/G C Grs16843804 44043 C/T C T rs2024115 44221 G/A A G rs10015979 49095 A/G AG rs7691627 51063 A/G G A rs2798235 54485 G/A G A rs4690072 62160 G/T TG rs6446723 66466 C/T T C rs363081 73280 G/A G A rs363080 73564 T/C C Trs363075 77327 G/A G A rs363064 81063 T/C C T rs3025849 83420 A/G A Grs6855981 87929 A/G G A rs363102 88669 G/A A G rs11731237 91466 C/T C Trs4690073 99803 A/G G A rs363144 100948 T/G T G rs3025838 101099 C/T C Trs34315806 101687 A/G G A rs363099 101709 T/C C T rs363096 119674 T/C TC rs2298967 125400 C/T T C rs2298969 125897 A/G G A rs6844859 130139 C/TT C rs363092 135682 C/A C A rs7685686 146795 A/G A G rs363088 149983 A/TA T rs362331 155488 C/T T C rs916171 156468 G/C c G rs362322 161018 A/GA G rs362275 164255 T/C C T rs362273 167080 A/G A G rs2276881 171314 G/AG A rs3121419 171910 T/C C T rs362272 174633 G/A G A rs362271 175171 G/AG A rs3775061 178407 C/T C T rs362310 179429 A/G G A rs362307 181498 T/CC T rs362306 181753 G/A G A rs362303 181960 T/C C T rs362296 186660 C/AC A rs1006798 198026 A/G A G

Example 41 Modified Oligonucleotides Targeting Huntingtin (HTT) SingleNucleotide Polymorphism (SNP)

A series of modified oligonucleotides were designed based on the parentgapmer, ISIS 460209 wherein the central gap region contains nine2′-deoxyribonucleosides. These modified oligonucleotides were designedby introducing various chemical modifications in the central gap regionand were tested for their ability to selectively inhibit mutant (mut)HTT mRNA expression levels targeting rs7685686 while leaving theexpression of the wild-type (wt) intact. The activity and selectivity ofthe modified oligonucleotides were evaluated and compared to the parentgapmer, ISIS 460209.

The modified oligonucleotides were created with a 3-9-3 motif and aredescribed in Table 57. The internucleoside linkages throughout eachgapmer are phosphorothioate (P═S) linkages. All cytosine nucleobasesthroughout each gapmer are 5-methyl cytosines. Nucleosides without asubscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by asubscript “e”, “k”, “y”, or “z” are sugar modified nucleosides. Asubscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, asubscript “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt), asubscript “y” indicates an α-L-LNA bicyclic nucleoside and a subscript“z” indicates a F-HNA modified nucleoside. ^(p)U indicates a 5-propyneuridine nucleoside and ^(x)T indicates a 2-thio-thymidine nucleoside.

The number in parentheses indicates the position on the modifiedoligonucleotide opposite to the SNP position, as counted from the5′-terminus.

Cell Culture and Transfection

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used (from Coriell Institute). CulturedGM04022 cells at a density of 25,000 cells per well were transfectedusing electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrationsof modified oligonucleotides. After a treatment period of approximately24 hours, cells were washed with DPBS buffer and lysed. RNA wasextracted using Qiagen RNeasy purification and mRNA levels were measuredby quantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. RT-PCR method in short; A mixture was madeusing 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uLwater and 40.4 uL RT MIX. To each well was added 15 uL of this mixtureand 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels weremeasured simultaneously by using two different fluorophores, FAM formutant allele and VIC for wild-type allele. The HTT mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN andthe results are presented below.

Analysis of IC₅₀'s

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis presented in Table 58 and was calculated by plotting theconcentrations of oligonucleotides used versus the percent inhibition ofHTT mRNA expression achieved at each concentration, and noting theconcentration of oligonucleotide at which 50% inhibition of HTT mRNAexpression was achieved compared to the control. The IC₅₀ at which eacholigonucleotide inhibits the mutant HTT mRNA expression is denoted as‘mut IC₅₀’. The IC₅₀ at which each oligonucleotide inhibits thewild-type HTT mRNA expression is denoted as ‘wt IC₅₀’. Selectivity wascalculated by dividing the IC₅₀ for inhibition of the wild-type HTTversus the IC₅₀ for inhibiting expression of the mutant HTT mRNA.

The parent gapmer, ISIS 460209 is marked with an asterisk (*) in thetable and was included in the study as a benchmark oligonucleotideagainst which the activity and selectivity of the modifiedoligonucleotides targeting nucleotides overlapping the SNP positioncould be compared.

As illustrated in Table 58, modified oligonucleotides having chemicalmodifications in the central gap region at the SNP position exhibitedsimilar activity with an increase in selectivity comparing to the parentgapmer, wherein the central gap region contains fulldeoxyribonucleosides.

TABLE 57 Modified oligonucleotides targeting HTT rs7685686 Wing SEQ ISISGap chemistry ID NO Sequence (5′ to 3′) chemistry 5′ 3′ NO 460209* (8)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) Full Deoxy ekk kke 10 539560 (8)T_(e)A_(k)A_(k)ATTG^(p)UCATCA_(k)C_(k)C_(e) Deoxy/5-Propyne ekk kke 11539563 (8) T_(e)A_(k)A_(k)ATTG^(x)TCATCA_(k)C_(k)C_(e) Deoxy/2-Thio ekkkke 10 539554 (8) T_(e)A_(k)A_(k)ATTGU_(y)CATCA_(k)C_(k)C_(e)Deoxy/α-L-LNA ekk kke 11 542686 (8)T_(e)A_(k)A_(k)ATTGT_(z)CATCA_(k)C_(k)C_(e) Deoxy/F-HNA ekk kke 10 e =2′-MOE, k = cEt

TABLE 58 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeted to rs7685686 inGM04022 cells ISIS Mut IC₅₀ Wt IC₅₀ Selectivity Wing chemistry NO (μM)(μM) (mut vs wt) Gap chemistry 5′ 3′  460209* (8) 0.41 2.0 4.9 FullDeoxy ekk kke 539560 (8) 0.29 1.1 3.8 Deoxy/5-Propyne ekk kke 539563 (8)0.45 3.1 6.9 Deoxy/2-Thio ekk kke 539554 (8) 3.5 >10 >3 Deoxy/α-L-LNAekk kke 542686 (8) 0.5 3.1 6.0 Deoxy/F-HNA ekk kke

Example 42 Modified Oligonucleotides Comprising Chemical Modificationsin the Gap Region Targeting Huntingtin (HTT) Single NucleotidePolymorphism (SNP)

Additional modified oligonucleotides were designed in a similar manneras the antisense oligonucleotides described in Table 57. Variouschemical modifications were introduced in the central gap region at theSNP position in an effort to improve selectivity while maintainingactivity in reducing mutant HTT mRNA levels.

The modified oligonucleotides were created with a 3-9-3 motif and aredescribed in Table 59. The internucleoside linkages throughout eachgapmer are phosphorothioate (P═S) linkages. All cytosine nucleobasesthroughout each gapmer are 5-methyl cytosines. Nucleosides without asubscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by asubscript “a”, “e”, “f”, “h”, “k”, “1”, “R”, “w” are sugar modifiednucleosides. A subscript “a” indicates a 2′-(ara)-F modified nucleoside,a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside,a subscript “f” indicates a 2′-F modified nucleoside, a subscript “h”indicates a HNA modified nucleoside, a subscript “k” indicates a6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt), a subscript “1” indicates aLNA modified nucleoside, a subscript “R” indicates a 5′-(R)-Me DNA, asubscript “w” indicates an unlocked nucleic acid (UNA) modifiednucleoside. ^(n)T indicates an N3-ethylcyano thymidine nucleoside and^(b)N indicates an abasic nucleoside (e.g. 2′-deoxyribonucleosidecomprising a H in place of a nucleobase). Underlined nucleoside or thenumber in parentheses indicates the position on the modifiedoligonucleotide opposite to the SNP position, as counted from the5′-terminus.

Thermal Stability Assay

The modified oligonucleotides were evaluated in thermal stability(T_(m)) assay. The T_(m)'s were measured using the method describedherein. A Cary 100 Bio spectrophotometer with the Cary Win UV Thermalprogram was used to measure absorbance vs. temperature. For the T_(m)experiments, oligonucleotides were prepared at a concentration of 8 μMin a buffer of 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, pH 7.Concentration of oligonucleotides were determined at 85° C. Theoligonucleotide concentration was 4 μM with mixing of equal volumes oftest oligonucleotide and mutant or wild-type RNA strand.Oligonucleotides were hybridized with the mutant or wild-type RNA strandby heating duplex to 90° C. for 5 min and allowed to cool at roomtemperature. Using the spectrophotometer, T_(m) measurements were takenby heating duplex solution at a rate of 0.5 C/min in cuvette starting @15° C. and heating to 85° C. T_(m) values were determined using VantHoff calculations (A₂₆₀ vs temperature curve) using nonself-complementary sequences where the minimum absorbance which relatesto the duplex and the maximum absorbance which relates to the non-duplexsingle strand are manually integrated into the program.

Presented in Table 60 is the T_(m) for the modified oligonucleotideswhen duplexed to mutant or wild-type RNA complement. The T_(m) of themodified oligonucleotides duplexed with mutant RNA complement is denotedas “T_(m) (° C.) mut”. The T_(m) of the modified oligonucleotidesduplexed with wild-type RNA complement is denoted as “T_(m) (° C.) wt”.

Cell Culture, Transfection and Selectivity Analysis

The modified oligonucleotides were also tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith a single dose at 2 μM concentration of the modifiedoligonucleotide. After a treatment period of approximately 24 hours,cells were washed with DPBS buffer and lysed. RNA was extracted usingQiagen RNeasy purification and mRNA levels were measured by quantitativereal-time PCR using ABI assay C_(—)2229297_(—)10 which measures at dbSNPrs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCRbuffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RTMIX. To each well was added 15 uL of this mixture and 5 uL of purifiedRNA. The mutant and wild-type HTT mRNA levels were measuredsimultaneously by using two different fluorophores, FAM for mutantallele and VIC for wild-type allele. The HTT mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN. The results inTable 60 are presented as percent of HTT mRNA expression, relative tountreated control levels and is denoted as “% UTC”. Selectivity as wasalso evaluated and measured by dividing the percent of wild-type HTTmRNA levels vs. the percent of mutant HTT mRNA levels.

The parent gapmer, ISIS 460209 is marked with an asterisk (*) in thetable and was included in the study as a benchmark oligonucleotideagainst which the selectivity of the modified oligonucleotides targetingnucleotides overlapping the SNP position could be compared.

As illustrated in Table 60, improvement in selectivity was observed forantisense oligonucleotides comprising chemical modifications in thecentral gap region at the SNP site such as 5′-(R)-Me (ISIS 539558), HNA(ISIS 539559), and 2′-(ara)-F (ISIS 539565) in comparison to the parentfull deoxy gapmer, ISIS 460209. Modified oligonucleotides comprising LNA(ISIS 539553) or 2′-F (ISIS 539570) showed comparable selectivity whileUNA modification (ISIS 539556 or 543909) showed no selectivity. Modifiedoligonucleotides comprising modified nucleobase, N3-ethylcyano (ISIS539564) or abasic nucleobase (ISIS 543525) showed little to noimprovement in selectivity.

TABLE 59 Modified oligonucleotides comprising chemical modificationsin the central gap region Wing SEQ ISIS chemistry ID NO Sequence (5′to 3′) Gap chemistry 5′ 3′ NO. 460209* (8)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) Full Deoxy ekk kke 10 539553 (8)T_(e)A_(k)A_(k)ATTGT_(l) CATCA_(k)C_(k)C_(e) Deoxy/LNA ekk kke 10539556 (8) T_(e)A_(k)A_(k)ATTGU_(w) CATCA_(k)C_(k)C_(e) Deoxy/UNA ekkkke 11 539558 (8) T_(e)A_(k)A_(k)ATTGT_(R) CATCA_(k)C_(k)C_(e)Deoxy/5′-(R)-Me DNA ekk kke 10 539559 (8) T_(e)A_(k)A_(k)ATTGT_(h)CATCA_(k)C_(k)C_(e) Deoxy/HNA ekk kke 10 539564 (8) T_(e)A_(k)A_(k)ATTG^(n)TCATCA_(k)C_(k)C_(e) Deoxy/deoxy with N3- ekk kke 10Ethylcyano nucleobase 539565 (8) T_(e)A_(k)A_(k)ATTGT_(a)CATCA_(k)C_(k)C_(e) Deoxy/2′-(ara)-F ekk kke 10 539570 (8)T_(e)A_(k)A_(k)ATTGT_(f) CATCA_(k)C_(k)C_(e) Deoxy/2′-F ekk kke 10543525 (8) T_(e)A_(k)A_(k)ATTG ^(b)NCATCA_(k)C_(k)C_(e)Deoxy/Deoxy-Abasic ekk kke 12 543909 (5) T_(e)A_(k)A_(k)AU_(w)TGTCATCA_(k)C_(k)C_(e) Deoxy/UNA ekk kke 13 e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

TABLE 60 Comparison of selectivity in inhibition of HTT mRNA levels andTm of modified oligonucleotides with ISIS 460209 targeted to rs7685686in GM04022 cells Wing ISIS Tm (° C.) % UTC Selectivity chemistry NOmutant wt mutant wt (wt vs mut) Gap chemistry 5′ 3′  460209* (8) 53.752.2 23 57 2.4 Full Deoxy ekk kke 539553 (8) 57.7 55.3 54 102 1.9Deoxy/LNA ekk kke 539556 (8) 43.7 44.1 90 105 1.2 Deoxy/UNA ekk kke539558 (8) 51.2 49.7 25 83 3.3 Deoxy/5′-(R)-Me DNA ekk kke 539559 (8)55.4 50.5 18 62 3.5 Deoxy/HNA ekk kke 539564 (8) 42.8 43.1 86 135 1.6Deoxy/Deoxy N3- ekk kke ethylcyano nucleobase 539565 (8) 53.8 52.5 14 463.4 Deoxy/2′-(ara)-F ekk kke 539570 (8) 54.4 51.8 25 50 2.0 Deoxy/2′-Fekk kke 543525 (8) 43.1 43.8 87 97 1.1 Deoxy/Deoxy Abasic ekk kke 543909(5) 44.7 42.1 68 79 1.2 Deoxy/UNA ekk kke e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

Example 43 Chimeric Oligonucleotides Comprising Self-ComplementaryRegions Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

Chimeric oligonucleotides were designed based on the parent gapmer, ISIS460209. These gapmers comprise self-complementary regions flanking thecentral gap region, wherein the central gap region contains ninedeoxyribonucleosides and the self-complementary regions arecomplementary to one another. The underlined nucleosides indicate theportion of the 5′-end that is self-complement to the portion of the3′-end.

The gapmers and their motifs are described in Table 61. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt).

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith a single dose at 2 μM concentration of the modifiedoligonucleotide. After a treatment period of approximately 24 hours,cells were washed with DPBS buffer and lysed. RNA was extracted usingQiagen RNeasy purification and mRNA levels were measured by quantitativereal-time PCR using ABI assay C_(—)2229297_(—)10 which measures at dbSNPrs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCRbuffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RTMIX. To each well was added 15 uL of this mixture and 5 uL of purifiedRNA. The mutant and wild-type HTT mRNA levels were measuredsimultaneously by using two different fluorophores, FAM for mutantallele and VIC for wild-type allele. HTT mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN. The results inTable 62 are presented as percent of HTT mRNA expression, relative tountreated control levels and is denoted as “% UTC”. Selectivity was alsoevaluated and measured by dividing the percent of wild-type HTT mRNAlevels vs. the percent of the mutant HTT mRNA levels.

The parent gapmer, ISIS 460209 is marked with an asterisk (*) in thetable and was included in the study as a benchmark oligonucleotideagainst which the selectivity of the modified oligonucleotides targetingnucleotides overlapping the SNP position could be compared.

As illustrated in Table 62, improvement in selectivity was observed forchimeric oligonucleotides comprising 5-9-5 (ISIS 550913), 6-9-6 (ISIS550912), 6-9-3 (ISIS 550907) or 3-9-7 (ISIS 550904) in comparison to theparent gapmer motif, 3-9-3 (ISIS 460209). The remaining gapmers showedmoderate to little improvement in selectivity.

TABLE 61 Chimeric oligonucleotides comprising various wing motifs targeted to HTT rs7685686 Wing SEQ ISIS chemistry ID NO Sequence (5′to 3′) Motif 5′ 3′ NO. 460209* T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)3-9-3 ekk kke 10 544838 T_(e) A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) A_(k)3-9-4 ekk kkek 14 544840 T_(e)A_(k)A_(k) ATTGTCATCA_(k)C_(k)C_(e)T_(k)T_(k)A_(k) 3-9-6 ekk kkekkk 15 544842T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) A_(k)T_(k)T_(k)T_(k)A_(k) 3-9-8ekk kkekkkkk 16 550903 T_(e)A_(k) A_(k)ATTGTCATCA_(k)C_(k)C_(e)T_(k)A_(k) 3-9-5 ekk kkekk 17 550904T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) T_(k)T_(k)T_(k)A_(k) 3-9-7 ekkkkekkkk 18 550905 G_(k) T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k) C_(e) 4-9-3kekk kke 19 550906 G_(k)G_(k) T_(e)A_(k)A_(k)ATTGTCATCA_(k) C_(k)C_(e)5-9-3 kkekk kke 20 550907 G_(k)G_(k)T_(k)T_(e)A_(k)A_(k)ATTGTCATCA_(k)G_(k)C_(e) 6-9-3 kkkekk kke 21 550908G_(k)G_(k)T_(k)G_(k) T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) 7-9-3kkkkekk kke 22 550909 G_(k)G_(k)T_(k)G_(k)A_(k)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) 8-9-3  kkkkkekk kke 23 550910G_(k)G_(k)C_(k) T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) G_(k)C_(k)C_(k)6-9-6 kkkekk kkekkk 24 550911 G_(k)C_(k)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) G_(k)C_(k) 5-9-5 kkekk kkekk 25550912 T_(k)A_(k)A_(k) T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)T_(k)T_(k)A_(k) 6-9-6 kkkekk kkekkk 26 550913 A_(k)A_(k)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) T_(k)T_(k) 5-9-5 kkekk kkekk 27550914 T_(k)C_(k)T_(k) T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)A_(k)G_(k)A_(k) 6-9-6 kkkekk kkekkk 28 550915 C_(k)T_(k)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) A_(k)G_(k) 5-9-5 kkekk kkekk 29e = 2′-MOE, k = cEt

TABLE 62 Comparison of selectivity in inhibition of HTT mRNA levels ofchimeric oligonucleotides with ISIS 460209 targeted to rs7685686 inGM04022 cells ISIS % UTC Selectivity wing chemistry NO mut wt (wt vs.mut) Motif 5′ 3′  460209* 23 57 2.4 3-9-3 ekk kke 544838 13 25 2.0 3-9-4ekk kkek 544840 17 31 1.8 3-9-6 ekk kkekkk 544842 55 102 1.9 3-9-8 ekkkkekkkkk 550903 13 36 2.7 3-9-5 ekk kkekk 550904 23 67 3.0 3-9-7 ekkkkekkkk 550905 21 51 2.4 4-9-3 kekk kke 550906 23 67 2.9 5-9-3 kkekk kke550907 30 93 3.1 6-9-3 kkkekk kke 550908 60 80 2.4 7-9-3 kkkkekk kke550909 42 101 2.4 8-9-3 kkkkkekk kke 550910 57 102 1.8 6-9-6 kkkekkkkekkk 550911 18 40 2.2 5-9-5 kkekk kkekk 550912 14 51 3.6 6-9-6 kkkekkkkekkk 550913 8 36 4.5 5-9-5 kkekk kkekk 550914 29 45 1.5 6-9-6 kkkekkkkekkk 550915 13 28 2.1 5-9-5 kkekk kkekk e = 2′-MOE, k = cEt

Example 44 Chimeric Antisense Oligonucleotides ComprisingNon-Self-Complementary Regions Targeting Huntingtin (HTT) SingleNucleotide Polymorphism (SNP)

Additional gapmers are designed based on the most selective gapmers fromstudies described in Tables 61 and 62 (ISIS 550912 and 550913). Thesegapmers are created such that they cannot form self-structure in theeffort to evaluate if the increased activity simply is due to higherbinding affinity. Gapmers are designed by deleting two or threenucleotides at the 3′-terminus and are created with 6-9-3 or 5-9-3motif.

The chimeric oligonucleotides and their motifs are described in Table63. The internucleoside linkages throughout each gapmer arephosphorothioate (P═S) linkages. All cytosine nucleobases throughouteach gapmer are 5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt).

The gapmers, ISIS 550912 and ISIS 550913, from which the newly designedgapmers are derived from, are marked with an asterisk (*) in the table.

TABLE 63 Non-self-complementary chimeric oligonucleotides  targeting HTT SNP Wing SEQ ISIS chemistry ID NO Sequence (5′ to 3′)Motif 5′ 3′ NO. 550912*T_(k)A_(k)A_(k)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)T_(k)T_(k)A_(k)6-9-6 kkkekk kkekkk 26 550913*A_(k)A_(k)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)T_(k)T_(k) 5-9-5 kkekkkkekk 27 556879 T_(k)A_(k)A_(k)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)6-9-3 kkkekk kke 30 556880A_(k)A_(k)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) 5-9-3 kkekk kke 31 e =2′-MOE, k = cEt

Example 45 Chimeric Oligonucleotides Containing Mismatches TargetingHuntingtin (HTT) Single Nucleotide Polymorphism (SNP)

A series of chimeric antisense oligonucleotides were designed based onthe parent gapmer, ISIS 460209, wherein the central gap region containsnine 2′-deoxyribonucleosides. These gapmers were designed by introducingmodified nucleosides at both 5′ and 3′ termini. Gapmers were alsocreated with a single mismatch shifted slightly upstream and downstream(i.e. “microwalk”) within the central gap region and with the SNPposition opposite position 5 of the parent gapmer, as counted from the5′-gap terminus.

The gapmers and their motifs are described in Table 64. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt). Underlinednucleosides indicate the mismatch position, as counted from the 5′-gapterminus.

These gapmers were evaluated for thermal stability (T_(m)) using methodsdescribed in Example 42. Presented in Table 65 are the T_(m)measurements for chimeric antisense oligonucleotides when duplexed tomutant or wild-type RNA complement. The T_(m) of chimeric antisenseoligonucleotides duplexed with mutant RNA complement is denoted as“T_(m) (° C.) mut”. The T_(m) of chimeric antisense oligonucleotidesduplexed with wild-type RNA complement is denoted as “T_(m) (° C.) wt”.

These gapmers were also tested in vitro. Heterozygous fibroblast GM04022cell line was used. Cultured GM04022 cells at a density of 25,000 cellsper well were transfected using electroporation with a single dose at 2μM concentration of the modified oligonucleotide. After a treatmentperiod of approximately 24 hours, cells were washed with DPBS buffer andlysed. RNA was extracted using Qiagen RNeasy purification and mRNAlevels were measured by quantitative real-time PCR using ABI assayC_(—)2229297_(—)10 which measures at dbSNP rs362303. RT-PCR method inshort; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers(300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well wasadded 15 uL of this mixture and 5 uL of purified RNA. The mutant andwild-type HTT mRNA levels were measured simultaneously by using twodifferent fluorophores, FAM for mutant allele and VIC for wild-typeallele. HTT mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN. The results in Table 65 are presented as percentof HTT mRNA expression, relative to untreated control levels and isdenoted as “% UTC”. Selectivity was also evaluated and measured bydividing the percent of wild-type HTT mRNA levels vs. the percent ofmutant HTT mRNA levels.

The parent gapmer, ISIS 460209 is marked with an asterisk (*) in thetable and was included in the study as a benchmark oligonucleotideagainst which the selectivity of the modified oligonucleotides targetingnucleotides overlapping the SNP position could be compared.

As illustrated in Table 65, improvement in selectivity was observed forgapmers comprising a 4-9-4 motif with a central deoxy gap region (ISIS476333) or a single mismatch at position 8 within the gap region (ISIS543531) in comparison to the parent gapmer. The remaining gapmers showedmoderate to little improvement in selectivity.

TABLE 64 Chimeric oligonucleotides containing a single mismatch targeting mutant HTT SNP Wing SEQ ISIS Mismatch chemistry ID NOSequence (5′ to 3′) position Motif 5′ 3′ NO. 460209*T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) — 3-9-3 ekk kke 10 476333A_(e)T_(k)A_(e)A_(k)ATTGTCATCA_(k)C_(e)C_(k)A_(e) — 4-9-4 ekek keke 32543526 A_(e)T_(k)A_(e)A_(k)ATTCTCATCA_(k)C_(e)C_(k)A_(e) 4 4-9-4 ekekkeke 33 543527 A_(e)T_(k)A_(e)A_(k)ATAGTCATCA_(k)C_(e)C_(k)A_(e) 3 4-9-4ekek keke 34 543529 A_(e)T_(k)A_(e)A_(k)ATTGTGATCA_(k)C_(e)C_(k)A_(e) 64-9-4 ekek keke 35 543530A_(e)T_(k)A_(e)A_(k)ATTGTCTTCA_(k)C_(e)C_(k)A_(e) 7 4-9-4 ekek keke 36543531 A_(e)T_(k)A_(e)A_(k)ATTGTCAACA_(k)C_(e)C_(k)A_(e) 8 4-9-4 ekkkeke 37 543532 T_(e)A_(k)A_(k)ATTCTCATCA_(k)C_(k)C_(e) 4 3-9-3 ekk kke38 543534 T_(e)A_(k)A_(k)AATGTCATCA_(k)C_(k)C_(e) 2 3-9-3 ekk kke 39543535 T_(e)A_(k)A_(k)ATTGTGATCA_(k)C_(k)C_(e) 6 3-9-3 ekk kke 40 543536T_(e)A_(k)A_(k)ATTGTCTTCA_(k)C_(k)C_(e) 7 3-9-3 ekk kke 41 543537T_(e)A_(k)A_(k)ATTGTCAACA_(k)C_(k)C_(e) 8 3-9-3 ekk kke 42 e = 2′-MOE, k= cEt

TABLE 65 Comparison of selectivity and T_(m) of chimericoligonucleotides with ISIS 460209 targeted to rs7685686 in GM04022 cellsISIS Tm (° C.) % UTC Selectivity Mismatch Wing chemistry NO mut wt mutwt (wt vs mut) position Motif 5′ 3′  460209* 53.7 52.2 23 57 2.4 — 3-9-3ekk kke 476333 60.2 58.4 10 37 3.6 — 4-9-4 ekek keke 543526 47.9 46.6 7086 1.2 4 4-9-4 ekek keke 543527 52.6 49.9 40 103 2.6 3 4-9-4 ekek keke543529 50.3 49.0 66 102 1.5 6 4-9-4 ekek keke 543530 52.9 50.9 67 1101.6 7 4-9-4 ekek keke 543531 53.3 50.3 46 136 3.0 8 4-9-4 ekk keke543532 43.6 42.8 127 151 1.2 4 3-9-3 ekk kke 543534 45.9 43.8 67 95 1.42 3-9-3 ekk kke 543535 44.0 43.3 96 113 1.2 6 3-9-3 ekk kke 543536 46.844.6 106 104 1.0 7 3-9-3 ekk kke 543537 45.9 44.3 77 81 1.1 8 3-9-3 ekkkke e = 2′-MOE, k = cEt

Example 46 Chimeric Oligonucleotides Comprising Mismatches TargetingHuntingtin (HTT) Single Nucleotide Polymorphism (SNP)

Additional chimeric antisense oligonucleotides are designed based on twogapmers selected from studies described in Tables 64 and 65 (ISIS 476333and ISIS 460209) wherein the central gap region contains nine2′-deoxyribonucleosides. These gapmers are designed by introducing asingle mismatch, wherein the mismatch will be shifted throughout theantisense oligonucleotide (i.e. “microwalk”). Gapmers are also createdwith 4-9-4 or 3-9-3 motifs and with the SNP position opposite position 8of the original gapmers, as counted from the 5′-terminus.

The gapmers and their motifs are described in Table 66. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt). Underlinednucleosides indicate the mismatch position, as counted from the5′-terminus.

The gapmers, ISIS 476333 and ISIS 460209, in which the newly designedantisense oligonucleotides are derived from, are marked with an asterisk(*) in the table.

TABLE 66 Chimeric oligonucleotides comprising mismatches targeting HTT SNP Wing SEQ ISIS Mismatch chemistry ID NO Sequence (5′to 3′) position Motif 5′ 3′ NO 476333*A_(e)T_(k)A_(e)A_(k)ATTGTCATCA_(k)C_(e)C_(k)A_(e) — 4-9-4 ekek keke 32554209 T_(e) T_(k)A_(e)A_(k)ATTGTCATCA_(k)C_(e)C_(k)A_(e)  1 4-9-4 ekekkeke 43 554210 A_(e) A_(k) A_(e)A_(k)ATTGTCATCA_(k)C_(e)C_(k)A_(e)  24-9-4 ekek keke 44 554211 A_(e)T_(k) T_(e)A_(k)ATTGTCATCA_(k)C_(e)C_(k)A_(e)  3 4-9-4 ekek keke 45 554212A_(e)T_(k)A_(e) T_(k) ATTGTCATCA_(k)C_(e)C_(k)A_(e)  4 4-9-4 ekek keke46 554213 A_(e)T_(k)A_(e)A_(k) TTTGTCATCA_(k)C_(e)C_(k)A_(e)  5 4-9-4ekek keke 47 554214 A_(e)T_(k)A_(e)A_(k)ATTGTCATGA_(k)C_(e)C_(k)A_(e) 134-9-4 ekek keke 48 554215 A_(e)T_(k)A_(e)A_(k)ATTGTCATCT_(k)C_(e)C_(k)A_(e) 14 4-9-4 ekek keke 49 554216A_(e)T_(k)A_(e)A_(k)ATTGTCATCA_(k) G_(e) C_(k)A_(e) 15 4-9-4 ekek keke50 554217 A_(e)T_(k)A_(e)A_(k)ATTGTCATCA_(k)C_(e) G_(k) A_(e) 16 4-9-4ekek keke 51 554218 A_(e)T_(k)A_(e)A_(k)ATTGTCATCA_(k)C_(e)C_(k) T_(e)17 4-9-4 ekek keke 52 460209* T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) —3-9-3 ekk kke 10 562481 T_(e)A_(k)A_(k) GTTGTCATCA_(k)C_(k)C_(e)  43-9-3 ekk kke 53 554482 T_(e)A_(k)A_(k)AGTGTCATCA_(k)C_(k)C_(e)  5 3-9-3ekk kke 54 554283 T_(e)A_(k)A_(k)ATGGTCATCA_(k)C_(k)C_(e)  6 3-9-3 ekkkke 55 e = 2′-MOE, k = cEt

Example 47 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin(HTT) Single Nucleotide Polymorphism (SNP)

Chimeric antisense oligonucleotides were designed based on the parentgapmer, ISIS 460209, wherein the central gap region contains nine2′-deoxyribonucleosides. These gapmers were designed by shortening thecentral gap region to seven 2′-deoxyribonucleosides. Gapmers were alsocreated with 5-7-5 motif and with the SNP position opposite position 8or 9 of the parent gapmer, as counted from the 5′-terminus.

The gapmers and their motifs are described in Table 67. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt). Underlinednucleoside or the number in parentheses indicates the position on themodified oligonucleotide opposite to the SNP position, as counted fromthe 5′-terminus.

The chimeric antisense oligonucleotides were tested in vitro. ISIS141923 was included in the study as a negative control and is denoted as“neg control”. A non-allele specific antisense oligonucleotide, ISIS387916 was used as a positive control and is denoted as “pos control”.ISIS 460209 was included in the study for comparison. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.12, 0.37, 1.1, 3.3, and 10 μM concentration of the modifiedoligonucleotide. After a treatment period of approximately 24 hours,cells were washed with DPBS buffer and lysed. RNA was extracted usingQiagen RNeasy purification and mRNA levels were measured by quantitativereal-time PCR using ABI assay C_(—)2229297_(—)10 which measures at dbSNPrs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCRbuffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RTMIX. To each well was added 15 uL of this mixture and 5 uL of purifiedRNA. The mutant and wild-type HTT mRNA levels were measuredsimultaneously by using two different fluorophores, FAM for mutantallele and VIC for wild-type allele. HTT mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN and the resultsare presented in Table 68.

The IC₅₀ and selectivity were calculated using methods describedpreviously in Example 41. As illustrated in Table 68, no improvement inpotency and selectivity was observed for the chimeric antisenseoligonucleotides as compared to ISIS 460209.

TABLE 67 Chimeric antisense oligonucleotides targeting HTT rs7685686Wing SEQ ISIS Chemistry ID NO Sequence (5′ to 3′) Motif 5′ 3′ NO.460209* (8) T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) 3-9-3 ekk kke 10460085 (9) A_(e)T_(e)A_(e)A_(e)A_(e)TTGTCATC_(e)A_(e)C_(e)C_(e)A_(e)5-7-5 eeeee eeeee 32 540108 (9)A_(e)T_(e)A_(e)A_(k)A_(k)TTGTCATC_(k)A_(k)C_(e)C_(e)A_(e) 5-7-5 eeekkkkeee 32 387916T_(e)C_(e)T_(e)C_(e)T_(e)ATTGCACATTC_(e)C_(e)A_(e)A_(e)G_(e) 5-10-5eeeee eeeee 56 (pos control) 141923C_(e)C_(e)T_(e)T_(e)C_(e)CCTGAAGGTTC_(e)C_(e)T_(e)C_(e)C_(e) 5-10-5eeeee eeeee 57 (neg control) e = 2′-MOE, k = cEt

TABLE 68 Comparison of inhibition of HTT mRNA levels and selectivity ofchimeric antisense oligonucleotides with ISIS 460209 targeted tors7685686 in GM04022 cells Wing Mut IC₅₀ Wt IC₅₀ Selectivity chemistryISIS NO (μM) (μM) (mut vs wt) Motif 5′ 3′ 460209* (8) 0.41 2.0 4.9 3-9-3ekk kke 460085 (9) 3.5 >10 >3 5-7-5 eeeee eeeee 540108 (9) 0.41 — —5-7-5 eeekk kkeee 387916 0.39 0.34 1.0 5-10-5 eeeee eeeee (pos control)141923 >10 >10 — 5-10-5 eeeee eeeee (neg control) e = 2′-MOE, k = cEt

Example 48 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin(HTT) Single Nucleotide Polymorphism (SNP)

Additional chimeric antisense oligonucleotides were designed based onthe parent gapmer, ISIS 460209, wherein the central gap region containsnine 2′-deoxyribonucleosides. These gapmers were designed with thecentral gap region shortened or interrupted by introducing variousmodifications either within the gap or by adding one or more modifiednucleosides to the 3′-most 5′-region or to the 5′-most 3′-region.Gapmers were created with the SNP position opposite position 8 of theparent gapmer, as counted from the 5′-terminus.

The gapmers and their motifs are described in Table 69. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt).

The chimeric antisense oligonucleotides were tested in vitro.Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022cells at a density of 25,000 cells per well were transfected usingelectroporation with 2 μM concentration of the modified oligonucleotide.After a treatment period of approximately 24 hours, cells were washedwith DPBS buffer and lysed. RNA was extracted using Qiagen RNeasypurification and mRNA levels were measured by quantitative real-time PCRusing ABI assay C_(—)2229297_(—)10 which measures at dbSNP rs362303.RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer,101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. Toeach well was added 15 uL of this mixture and 5 uL of purified RNA. Themutant and wild-type HTT mRNA levels were measured simultaneously byusing two different fluorophores, FAM for mutant allele and VIC forwild-type allele. HTT mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN. The results in Table 70 are presentedas percent of HTT mRNA expression, relative to untreated control levelsand is denoted as “% UTC”. Selectivity was also evaluated and measuredby dividing the percent of wild-type HTT mRNA levels vs. the percent ofmutant HTT mRNA levels. ISIS 460209 marked with an asterisk (*) in thetable was included in the study for comparison.

As illustrated in Table 70, modifications to the 3′-most 5′-regionnucleosides that shorten the gap from 9 to 7 or 8 nucleotides (ISIS551429 and ISIS 551426) improved selectivity and potency comparing tothe parent gapmer (ISIS 460209). The remaining chimeric antisenseoligonucleotides showed moderate to little improvement in selectivity.

TABLE 69 Short-gap antisense oligonucleotides targeting  HTT rs7685686Wing SEQ ISIS Chemistry ID NO Sequence (5′ to 3′) Motif 5′ 3′ NO.460209* T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) 3-9-3 ekk kke 10 551426T_(e)A_(k)A_(e)A_(k)TTGTCATCA_(k)C_(k)C_(e) 4-8-3 ekek kke 10 551427T_(e)A_(k)A_(e)AT_(k)TGTCATCA_(k)C_(k)C_(e) 3-9-3 or eke or kke 10 5-7-3ekedk 551428 T_(e)A_(k)A_(e)ATT_(k)GTCATCA_(k)C_(k)C_(e) 3-9-3 or eke orkke 10 6-6-3 ekeddk 551429T_(e)A_(e)A_(e)A_(k)T_(k)TGTCATCA_(k)C_(k)C_(e) 5-7-3 eeekk kke 10 e =2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

TABLE 70 Comparison of selectivity in inhition of HTT mRNA levels ofantisense oligonucleotides with ISIS 460209 targeted to rs7685686 inGM4022 cells % UTC Selectivity Wing chemistry ISIS NO mut wt (wt vs.mut) Motif 5′ 3′ 460209* 23 57 2.4 3-9-3 ekk kke 551426 14 66 4.8 4-8-3ekek kke 551427 35 97 2.8 3-9-3 or eke or kke 5-7-3 ekedk 551428 61 1101.8 3-9-3 or eke or kke 6-6-3 ekeddk 551429 19 94 5.0 5-7-3 eeekk kke e= 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 49 Modified Oligonucleotides Targeting HTT SNP

A series of modified antisense oligonucleotides are designed based onthe parent gapmer, ISIS 460209, wherein the central gap region containsnine 2′-deoxynucleosides and is marked with an asterisk (*) in thetable. These modified oligonucleotides are designed by shortening orinterrupting the gap with a single mismatch or various chemicalmodifications within the central gap region. The modifiedoligonucleotides are created with the SNP position opposite position 8of the parent gapmer, as counted from the 5′-terminus.

The gapmers and their motifs are described in Table 71. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages, except for the internucleoside linkage with a subscript“p”, “pz” or “pw”. Subscript “p” indicates methyl phosphonateinternucleoside linkage. Subscript “pz” indicates (R)-methyl phosphonateinternucleoside linkage. Subscript “pw” indicates (S)-methyl phosphonateinternucleoside linkage. All cytosine nucleobases throughout each gapmerare 5-methyl cytosines. ^(x)T indicates a 2-thio thymidine nucleoside.Nucleosides without a subscript are β-D-2′-deoxyribonucleosides.Nucleosides followed by a subscript “e”, “k” or “b” are sugar modifiednucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE)modified nucleoside, a subscript “k” indicates a 6′-(S)—CH₃ bicyclicnucleoside (e.g. cEt) and a subscript “b” indicates a 5′-Me DNA modifiednucleoside. Underlined nucleosides indicate the position ofmodification. Bold and underlined nucleosides indicate the mismatchposition.

TABLE 71 Short-gap chimeric oligonucleotides targeting  HTT SNP Wing SEQISIS Sequence Gap Chemistry ID NO (5′ to 3′) Motif Chemistry 5′ 3′ NO.460209* T_(e)A_(k)A_(k)ATTGTC 3-9-3 — ekk kke 10 ATCA_(k)C_(k)C_(e)XXXX16 T_(e)A_(k)A_(k)A ^(x)TTGT 3-9-3 Deoxy/2-thio ekk kke 10CATCA_(k)C_(k)C_(e) XXXX17 T_(e)A_(k)A_(k)AT ^(x)TGT 3-9-3 Deoxy/2-thioekk kke 10 CATCA_(k)C_(k)C_(e) XXXX18 T_(e)A_(k)A_(k)A ^(x)T^(x)TGT3-9-3 Deoxy/2-thio ekk kke 10 CATCA_(k)C_(k)C_(e) XXXX19T_(e)A_(k)A_(k)ATT_(p) GT 3-9-3 Deoxy/Methyl ekk kke 10 (558257)CATCA_(k)C_(k)C_(e) phosphonate XXXX20 T_(e)A_(k)A_(k)AT_(p) TGT 3-9-3Deoxy/Methyl ekk kke 10 (558256) CATCA_(k)C_(k)C_(e) phosphonate XXXX20aT_(e)A_(k)A_(k)AT_(pz) TGT 3-9-3 Deoxy/(R)- ekk kke 10CATCA_(k)C_(k)C_(e) Methyl phosphonate XXXX20b T_(e)A_(k)A_(k)AT_(pw) TG3-9-3 Deoxy/(S)- ekk kke 10 TCATCA_(k)C_(k)C_(e) Methyl phosphonateXXXX21 T_(e)A_(k)A_(k) A_(p) TTGT 3-9-3 Methyl ekk kke 10 (558255)CATCA_(k)C_(k)C_(e) phosphonate XXXX22 T_(e)A_(k)A_(k)ATT_(b) GT 3-9-35′-Me-DNA ekk kke 10 CATCA_(k)C_(k)C_(e) XXXX23 T_(e)A_(k)A_(k)AT_(b)TGT 3-9-3 5′-Me-DNA ekk kke 10 CATCA_(k)C_(k)C_(e) XXXX24T_(e)A_(k)A_(k) A_(b) TTGT 3-9-3 5′-Me-DNA ekk kke 10CATCA_(k)C_(k)C_(e) XXXX25 T_(e)A_(k)A_(k) G TTGTC 4-8-3 Mismatch at ekkkke 53 ATCA_(k)C_(k)C_(e) position 4 XXXX26 T_(e)A_(k)A_(k)A G TGT 5-7-3Mismatch at ekk kke 54 CATCA_(k)C_(k)C_(e) position 5 XXXX27T_(e)A_(k)A_(k)AT G GT 6-6-3 Mismatch at ekk kke 55 CATCA_(k)C_(k)C_(e)position 6 e = 2′-MOE, k = cEt

Example 50 Short-Gap Chimeric Oligonucleotides Comprising Modificationsat the Wing Regions Targeting Huntingtin (HTT) Single NucleotidePolymorphism (SNP)

Additional chimeric antisense oligonucleotides were designed based onthe parent gapmer, ISIS 460209, wherein the central gap region containsnine 2′-deoxynucleosides. These gapmers were designed by shortening thecentral gap region to seven 2′-deoxynucleosides and introducing variousmodifications at the wing regions.

The gapmers and their motifs are described in Table 72. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt).

The number in parentheses indicates the position on the chimericoligonucleotide opposite to the SNP position, as counted from the5′-terminus.

These gapmers were evaluated for thermal stability (T_(m)) using methodsdescribed in Example 42. Presented in Table 73 is the T_(m) measurementsfor chimeric antisense oligonucleotides when duplexed to mutant orwild-type RNA complement. The T_(m) of chimeric antisenseoligonucleotides duplexed with mutant RNA complement is denoted as“T_(m) (° C.) mut”. The T_(m) of chimeric antisense oligonucleotidesduplexed with wild-type RNA complement is denoted as “T_(m) (° C.) wt”.

These gapmers were also tested in vitro. Heterozygous fibroblast GM04022cell line was used. Cultured GM04022 cells at a density of 25,000 cellsper well were transfected using electroporation with a single dose at 2μM concentration of the modified oligonucleotide. After a treatmentperiod of approximately 24 hours, cells were washed with DPBS buffer andlysed. RNA was extracted using Qiagen RNeasy purification and mRNAlevels were measured by quantitative real-time PCR using ABI assayC_(—)2229297_(—)10 which measures at dbSNP rs362303. RT-PCR method inshort; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers(300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well wasadded 15 uL of this mixture and 5 uL of purified RNA. The mutant andwild-type HTT mRNA levels were measured simultaneously by using twodifferent fluorophores, FAM for mutant allele and VIC for wild-typeallele. HTT mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN. The results in Table 73 are presented as percentof HTT mRNA expression, relative to untreated control levels and isdenoted as “% UTC”. Selectivity was also evaluated and measured bydividing the percent of wild-type HTT mRNA levels vs. the percent ofmutant HTT mRNA levels. ISIS 460209 marked with an asterisk (*) in thetable was included in the study for comparison.

As illustrated in Table 73, improvement in selectivity was observed forgapmers comprising 2-7-8 or 5-7-5 motifs having cEt subunits at the wingregions in comparison to the parent gapmer, ISIS 460209. The remaininggapmers showed moderate to little improvement in selectivity.

TABLE 72 Short-gap chimeric oligonucleotides comprising  wing modifications wing SEQ ISIS chemistry ID NO Sequence (5′ to 3′)Motif 5′ 3′ NO. 460209* (8) T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)3-9-3 ekk kke 10 540103 (6)A_(k)A_(k)TTGTCATC_(e)A_(e)C_(e)C_(e)A_(e)G_(e)A_(e)A_(e) 2-7-8 kk e8 58540104(6)  A_(e)A_(e)TTGTCATC_(e)A_(e)C_(e)C_(e)A_(e)G_(e)A_(e)A_(e)2-7-8 ee e8 59 540105 (7)A_(e)A_(e)A_(e)TTGTCATC_(e)A_(e)C_(e)C_(e)A_(e)G_(e)A_(e) 3-7-7 eee e760 540106 (8) T_(e)A_(e)A_(e)A_(e)TTGTCATC_(e)A_(e)C_(e)C_(e)A_(e)G_(e)4-7-6 eeee e6 61 540107 (9)A_(e)T_(e)A_(e)A_(e)A_(k)TTGTCATC_(k)A_(e)C_(e)C_(e)A_(e) 5-7-5 eeeekkeeee 32 540109 (10)A_(e)A_(e)T_(e)A_(e)A_(e)A_(e)TTGTCATC_(e)A_(e)C_(e)C_(e) 6-7-4 e6 e4 62540110 (11) T_(e)A_(e)A_(e)T_(e)A_(e)A_(e)A_(e)TTGTCATC_(e)A_(e)C_(e)7-7-3 e7 eee 63 540111 (12)T_(e)T_(e)A_(e)A_(e)T_(e)A_(e)A_(e)A_(e)TTGTCATC_(e)A_(e) 8-7-2 e8 ee 64540112 (12) T_(e)T_(e)A_(e)A_(e)T_(e)A_(e)A_(e)A_(e)TTGTCATC_(k)A_(k)8-7-2 e8 kk 64 e = 2′-MOE (e.g. e6 = eeeeee), and k = cEt

TABLE 73 Comparison of selectivity in inhibition of HTT mRNA levels ofantisense oligonucleotides with ISIS 460209 targeted to RS7685686 inGM04022 cells Selec- Tm tivity wing (° C.) % UTC (wt vs chemistry ISISNO mut wt mut wt mut) Motif 5′ 3′ 460209* (8) 53.7 52.2 23 57 2.4 3-9-3ekk kke 540103 (6) 57.6 56.4 23 74 3.3 2-7-8 kk e8 540104 (6) 54.8 52.836 91 2.5 2-7-8 ee e8 540105 (7) 54.2 52.2 53 135 2.6 3-7-7 eee e7540106 (8) 52.4 50.8 30 77 2.6 4-7-6 eeee e6 540107 (9) 56.6 54.7 19 623.3 5-7-5 eeeek keeee 540109 (10) 49.1 47.3 78 127 1.6 6-7-4 e6 e4540110 (11) 42.8 41.2 89 112 1.3 7-7-3 e7 eee 540111 (12) 39.0 36.9 111128 1.1 8-7-2 e8 ee 540112 (12) 44.2 42.4 86 102 1.2 8-7-2 e8 kk

Example 51 Chimeric Oligonucleotides with SNP Site Shifting within theCentral Gap Region

Chimeric antisense oligonucleotides were designed based on the parentgapmer, ISIS 460209 wherein the SNP site aligns with position 5 of theparent gapmer, as counted from the 5′-gap terminus. These gapmers weredesigned by shifting the SNP site upstream or downstream (i.e.microwalk) within the central gap region of the parent gapmer.

The gapmers and their motifs are described in Table 74. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt). Underlinenucleosides indicate the position on the chimeric oligonucleotide alignswith the SNP site.

The SNP site indicates the position on the chimeric antisenseoligonucleotide opposite to the SNP position, as counted from the 5′-gapterminus and is denoted as “SNP site”.

The chimeric oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modifiedoligonucleotides. After a treatment period of approximately 16 hours,RNA was isolated from the cells and mRNA levels were measured byquantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. The HTT mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN. ISIS 460209 marked withan asterisk (*) in the table was included in the study for comparison.

The IC₅₀ and selectivity were calculated using the methods previouslydescribed in Example 41. As illustrated in Table 75, chimericoligonucleotides comprising 4-9-2 (ISIS 540082) or 2-9-4 (ISIS 540095)motif with the SNP site at position 1 or 3 showed comparable activityand 2.5 fold selectivity as compared to their counterparts.

TABLE 74 Chimeric oligonucleotides designed by microwalk wing SEQ ISISSNP chemistry ID NO Sequence (5′ to 3′) Motif site 5′ 3′ NO. 460209*T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) 3-9-3 5 ekk kke 10 540082A_(e)T_(k)T_(k)G_(k) TCATCACCAG_(k)A_(e) 4-9-2 1 ekkk ke 65 540089T_(e)T_(k)A_(k)A_(k)TAAATTGTCA_(k)T_(e) 4-9-2 8 ekkk ke 66 540095A_(e)T_(k)TGTCATCACC_(k)A_(k)G_(k)A_(e) 2-9-4 3 ek kkke 65 e = 2′-MOE,and k = cEt

TABLE 75 Comparison of inhibition of HTT mRNA levels and selectivity ofchimeric oligonucleotides with ISIS 460209 targeted to HTT SNP Mut WingIC₅₀ Wt IC₅₀ Selectivity SNP chemistry ISIS NO (μM) (μM) (wt vs mut)Motif site 5′ 3′ 460209 0.41 2.0 4.9 3-9-3 5 ekk kke 540082 0.45 5.6 124-9-2 1 ekkk ke 540089 >10 >10 — 4-9-2 8 ekkk ke 540095 0.69 8.4 122-9-4 3 ek kkke e = 2′-MOE, and k = cEt

Example 52 Chimeric Oligonucleotides with SNP Site Shifting at VariousPositions

Chimeric antisense oligonucleotides were designed based on the parentgapmer, ISIS 460209 wherein the SNP site aligns with position 8 of theparent gapmer, as counted from the 5′-terminus. These gapmers weredesigned by shifting the SNP site upstream or downstream (i.e.microwalk) of the original oligonucleotide.

The gapmers and their motifs are described in Table 76. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt). Underlinenucleosides indicate the SNP site.

The SNP site indicates the position on the chimeric antisenseoligonucleotide opposite to the SNP position, as counted from the5′-terminus and is denoted as “SNP site”.

The chimeric oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modifiedoligonucleotides. After a treatment period of approximately 16 hours,cells were washed with DPBS buffer and lysed. RNA was extracted usingQiagen RNeasy purification and mRNA levels were measured by quantitativereal-time PCR using ABI assay C_(—)2229297_(—)10 which measures at dbSNPrs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCRbuffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RTMIX. To each well was added 15 uL of this mixture and 5 uL of purifiedRNA. The mutant and wild-type HTT mRNA levels were measuredsimultaneously by using two different fluorophores, FAM for mutantallele and VIC for wild-type allele. HTT mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN. The results inTable 77 are presented as percent of HTT mRNA expression, relative tountreated control levels and is denoted as “% UTC”. Selectivity was alsoevaluated and measured by dividing the percent of wild-type HTT mRNAlevels vs. the percent of mutant HTT mRNA levels.

The parent gapmer, ISIS 460209 is marked with an asterisk (*) in thetable and was included in the study as a benchmark oligonucleotideagainst which the selectivity of the modified oligonucleotides targetingnucleotides overlapping the SNP position could be compared.

As illustrated in Table 77, improvement in potency and selectivity wasobserved for chimeric oligonucleotides comprising 4-9-2 or 2-9-4 motifhaving the target SNP site at positions 3, 4, 6, 7 and 8 (ISIS540083,ISIS540084, ISIS 540085, ISIS 540094, ISIS 540096, ISIS 540097 and ISIS540098) in comparison to position 8 of the parent gapmer (ISIS 460209).The remaining gapmers showed little to no improvement in potency orselectivity.

TABLE 76 Chimeric oligonucleotides designed by microwalk SEQ ISIS SNP IDNO Sequence (5′ to 3′) site Motif NO. 460209*T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)  8 3-9-3 10 (ekk- d9- kke)543887 T_(e)T_(k)G_(k) T_(k) CATCACCAGA_(k)A_(e)  4 4-9-2 67 (ekkk-d9-ke) 540083 A_(e)A_(k)T_(k)T_(k)GTCATCACCA_(k)G_(e)  6 4-9-2 68 (ekkk-d9-ke) 540084 A_(e)A_(k)A_(k)T_(k)TGTCATCACC_(k)A_(e)  7 4-9-2 69 (ekkk-d9-ke) 540085 T_(e)A_(k)A_(k)A_(k)TTGTCATCAC_(k)C_(e)  8 4-9-2 10 (ekkk-d9-ke) 540087 A_(e)A_(k)T_(k)A_(k)AATTGTCATC_(k)A_(e) 10 4-9-2 70 (ekkk-d9-ke) 540090 A_(e)T_(k)T_(k)A_(k)ATAAATTGTC_(k)A_(e) 13 4-9-2 71 (ekkk-d9-ke) 540091 T_(e)A_(k)T_(k)T_(k)AATAAATTGT_(k) C_(e) 14 4-9-2 72(ekkk- d9-ke) 540092 G_(e) T_(k) CATCACCAGA_(k)A_(k)A_(k)A_(e)  2 2-9-473 (ek- d9- kkke) 540093 T_(e)G_(k) TCATCACCAG_(k)A_(k)A_(k)A_(e)  32-9-4 74 (ek- d9- kkke) 540094 T_(e)T_(k)GTCATCACCA_(k)G_(k)A_(k)A_(e) 4 2-9-4 67 (ek- d9- kkke) 540096A_(e)A_(k)TTGTCATCAC_(k)C_(k)A_(k)G_(e)  6 2-9-4 68 (ek- d9- kkke)540097 A_(e)A_(k)ATTGTCATCA_(k)C_(k)C_(k)A_(e)  8 2-9-4 69 (ek- d9-kkke) 540098 T_(e)A_(k)AATTGTCATC_(k)A_(k)C_(k)C_(e)  8 2-9-4 10 (ek-d9- kkke) 540099 A_(e)T_(k)AAATTGTCAT_(k)C_(k)A_(k)C_(e)  9 2-9-4 75(ek- d9- kkke) 540100 A_(e)A_(k)TAAATTGTCA_(k)T_(k)C_(k)A_(e) 10 2-9-470 (ek- d9- kkke) 540101 T_(e)A_(k)ATAAATTGTC_(k)A_(k)T_(k)C_(e) 112-9-4 76 (ek- d9- kkke) 540102 T_(e)T_(k)AATAAATTGT_(k) C_(k)A_(k)T_(e)12 2-9-4 66 (ek- d9- kkke) e = 2′-MOE; k = cEt; d =2′-deoxyribonucleoside

TABLE 77 Comparison of selectivity in HTT SNP inhibition of chimericoligonucleotides with ISIS 460209 % UTC Selectivity SNP ISIS NO mut wt(wt vs. mut) site Motif  460209* 23 57 2.4 8 3-9-3 (ekk-d9-kke) 54388718 43 2.3 4 4-9-2 (ekkk-d9-ke) 540083 18 67 3.7 6 4-9-2 (ekkk-d9-ke)540084 10 49 4.9 7 4-9-2 (ekkk-d9-ke) 540085 21 86 4.1 8 4-9-2(ekkk-d9-ke) 540087 60 98 1.6 10 4-9-2 (ekkk-d9-ke) 540090 129 137 1.113 4-9-2 (ekkk-d9-ke) 540091 93 105 1.1 14 4-9-2 (ekkk-d9-ke) 540092 2855 2.0 2 2-9-4 (ek-d9-kkke) 540093 18 62 3.4 3 2-9-4 (ek-d9-kkke) 54009413 45 3.4 4 2-9-4 (ek-d9-kkke) 540096 17 68 4.0 6 2-9-4 (ek-d9-kkke)540097 8 35 4.2 8 2-9-4 (ek-d9-kkke) 540098 12 45 3.9 8 2-9-4(ek-d9-kkke) 540099 62 91 1.5 9 2-9-4 (ek-d9-kkke) 540100 80 106 1.3 102-9-4 (ek-d9-kkke) 540101 154 152 1.0 11 2-9-4 (ek-d9-kkke) 540102 102106 1.0 12 2-9-4 (ek-d9-kkke) e = 2′-MOE; k = cEt; d =2′-deoxyribonucleoside

Example 53 Selectivity in Inhibition of HTT mRNA Levels Targeting SNP byChimeric Oligonucleotides Designed by Microwalk

A series of modified oligonucleotides were designed based on the parentgapmer, ISIS 460209, wherein the central gap region comprises nine2′-deoxyribonucleosides. These gapmers were created with various motifsand modifications at the wings and/or the central gap region.

The modified oligonucleotides and their motifs are described in Table78. The internucleoside linkages throughout each gapmer arephosphorothioate (P═S) linkages. All cytosine nucleobases throughouteach gapmer are 5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”,“k”, “y”, or “z” are sugar modified nucleosides. A subscript “e”indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt), a subscript “y”indicates an α-L-LNA modified nucleoside, and a subscript “z” indicatesa F-HNA modified nucleoside. ^(p)U indicates a 5-propyne uridinenucleoside and ^(x)T indicates a 2-thio-thymidine nucleoside. Underlinednucleosides indicate the mismatch position.

These gapmers were evaluated for thermal stability (T_(m)) using methodsdescribed in Example 42. Presented in Table 79 are the T_(m)measurements for chimeric antisense oligonucleotides when duplexed tomutant or wild-type RNA complement. The T_(m) of chimeric antisenseoligonucleotides duplexed with mutant RNA complement is denoted as“T_(m) (° C.) mut”. The T_(m) of chimeric antisense oligonucleotidesduplexed with wild-type RNA complement is denoted as “T_(m) (° C.) wt”.

These gapmers were also tested in vitro. ISIS 141923 was included in thestudy as a negative control and is denoted as “neg control”. Thenon-allele specific antisense oligonucleotides, ISIS 387916 was used asa positive control and is denoted as “pos control”. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith a single dose at 2 μM concentration of the modifiedoligonucleotide. After a treatment period of approximately 24 hours,cells were washed with DPBS buffer and lysed. RNA was extracted usingQiagen RNeasy purification and mRNA levels were measured by quantitativereal-time PCR using ABI assay C_(—)2229297_(—)10 which measures at dbSNPrs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCRbuffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RTMIX. To each well was added 15 uL of this mixture and 5 uL of purifiedRNA. The mutant and wild-type HTT mRNA levels were measuredsimultaneously by using two different fluorophores, FAM for mutantallele and VIC for wild-type allele. HTT mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN. ISIS 460209marked with an asterisk (*) in the table was included in the study forcomparison. The results in Table 79 are presented as percent of HTT mRNAexpression, relative to untreated control levels and is denoted as “%UTC”. Selectivity was also evaluated and measured by dividing thepercent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNAlevels.

As illustrated, several of the newly designed antisense oligonucleotidesshowed improvement in potency and/or selectivity in inhibiting mut HTTmRNA levels comparing to ISIS 460209.

TABLE 78Modified oligonucleotides comprising various modifications targeting HTT SNP Wing SEQ ISIS Chemistry ID NO Sequence (5′ to 3′) Modification5′ 3′ NO. 460209* T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) 3-9-3 ekk kke10 (ekk-d9-kke) 539560 T_(e)A_(k)A_(k)ATTG^(p)UCATCA_(k)C_(k)C_(e)5-propyne in ekk kke 11 gap 539563T_(e)A_(k)A_(k)ATTG^(x)TCATCA_(k)C_(k)C_(e) 2-thio in gap ekk kke 10539554 T_(e)A_(k)A_(k)ATTGU_(y)CATCA_(k)C_(k)C_(e) α-L-LNA in gap ekkkke 11 542686 T_(e)A_(k)A_(k)ATTGT_(z)CATCA_(k)C_(k)C_(e) F-HNA in gapekk kke 10 540108A_(e)T_(e)A_(e)A_(k)A_(k)TTGTCATC_(k)A_(k)C_(e)C_(e)A_(e) 5-7-5 eeekkkkeee 23 (eeekk-d7-kkeee) 544840T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)T_(k)T_(k)A_(k) 3-9-6 ekk kkekkk15 (ekk-d9-kkekkk) 550904T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e)T_(k)T_(k)T_(k)A_(k) 3-9-7 ekkkkekkkk 18 (ekk-d9-kkekkkk) 540082A_(e)T_(k)T_(k)G_(k)TCATCACCAG_(k)A_(e) 4-9-2 ekkk ke 65 (ekkk-d9-ke)540089 T_(e)T_(k)A_(k)A_(k)TAAATTGTCA_(k)T_(e) 4-9-2 ekkk ke 66(ekkk-d9-ke) 540095 A_(e)T_(k)TGTCATCACC_(k)A_(k)G_(k)A_(e) 2-9-4 ekkkke 67 (ek-d9-kkke) 543528A_(e)T_(k)A_(e)A_(k)AATGTCATCA_(k)C_(e)C_(k)A_(e) Mismatch at ekek keke77 position 2  counting from 5′ gap 543533T_(e)A_(k)A_(k)ATAGTCATCA_(k)C_(k)C_(e) Mismatch at ekk kke 78position 3  counting from 5′ gap 387916T_(e)C_(e)T_(e)C_(e)T_(e)ATTGCACATTC_(e)C_(e)A_(e)A_(e)G_(e) 5-10-5eeeee eeeee 56 (pos control) 141923C_(e)C_(e)T_(e)T_(e)C_(e)CCTGAAGGTTC_(e)C_(e)T_(e)C_(e)C_(e) 5-10-5eeeee eeeee 57 (neg control) e = 2′-MOE; k = cEt; d =2′-deoxyribonucleoside

TABLE 79 Comparison of selectivity in inhibition of HTT mRNA levels, andTm of modified oligonucleotides with ISIS 460209 targeted tors7685686 inGM04022 cells Tm (° C.) % UTC Selectivity Wing Chemistry ISIS NO mutantwt mut wt (wt vs mut) Modification 5′ 3′ 460209* 53.7 52.2 23 57 2.73-9-3 ekk kke (ekk-d9-kke) 539560 54.1 50.8 13 32 2.4 5-propyne in gapekk kke 539563 53.8 49.1 13 40 3.2 2-thio in gap ekk kke 539554 56.554.5 54 89 1.7 α-L-LNA in gap ekk kke 542686 56.1 50.4 26 62 2.4 F-HNAin gap ekk kke 540108 60.0 57.9 27 63 2.3 5-7-5 eeekk kkeee(eeekk-d7-kkeee) 544840 — — 19 40 2.1 3-9-6 ekk kkekkk (ekk-d9-kkekkk)550904 — — 39 65 1.7 3-9-7 ekk kkekkkk (ekk-d9- kkekkkk) 540082 — — 2162 3.0 4-9-2 ekkk ke (ekkk-d9-ke) 540089 — — 78 86 1.1 4-9-2 ekkk ke(ekkk-d9-ke) 540095 — — 22 66 3.1 2-9-4 ek kkke (ek-d9-kkke) 543528 50.549.1 44 90 2.1 Mismatch at ekek keke position 2 counting from 5′ gap543533 47.0 44.8 83 97 1.2 Mismatch at ekk kke position 3 counting from5′ gap 387916 — — 21 19 0.9 5-10-5 eeeee eeeee (pos control) 141923 — —95 99 1.0 5-10-5 eeeee eeeee (neg control) e = 2′-MOE; k = cEt; d =2′-deoxyribonucleoside

Example 54 Chimeric Oligonucleotides Comprising Modifications at the SNPSite of HTT Gene

Additional gapmers are designed based on the gapmer selected fromstudies described in Tables 73 and 74 (ISIS 540108) and is marked withan asterisk (*). These gapmers are designed by introducing modificationsat the SNP site at position 9 of the oligonucleotides, as counted fromthe 5′-terminus and are created with a 5-7-5 motif.

The gapmers are described in Table 80. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytosinenucleobases throughout each gapmer are 5-methyl cytosines. Nucleosideswithout a subscript are β-D-2′-deoxyribonucleosides. Nucleosidesfollowed by a subscript “a”, “b”, “e”, or “k” are sugar modifiednucleosides. A subscript “a” indicates 2′-(ara)-F modified nucleoside, asubscript “b” indicates a 5′-Me DNA modified nucleoside, a subscript “e”indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, and a subscript“k” indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt). ^(x)Tindicates a 2-thio-thymidine nucleoside. Underline nucleoside or thenumber in parentheses indicates the position on the oligonucleotidesopposite to the SNP position, as counted from the 5′-terminus.

TABLE 80 Modified oligonucleotides targeting HTT SNP Wing SEQ ISIS Gapchemistry ID NO Sequence (5′ to 3′) Chemistry 5′ 3′ NO. 540108* (9)A_(e)T_(e)A_(e)A_(k)A_(k)TTGTCATC_(k)A_(k)C_(e)C_(e)A_(e) Deoxy eeekkkkeee 32 XXXX28 (9) A_(e)T_(e)A_(e)A_(k)A_(k)TTG^(x)TCATC_(k)A_(k)C_(e)C_(e)A_(e) Deoxy/2- eeekk kkeee 32 thioXXXX29 (9) A_(e)T_(e)A_(e)A_(k)A_(k)TTGT_(a)CATC_(k)A_(k)C_(e)C_(e)A_(e) Deoxy/2′- eeekk kkeee 32 (ara)-F XXXX30 (9)A_(e)T_(e)A_(e)A_(k)A_(k)TTGT_(b) CATC_(k)A_(k)C_(e)C_(e)A_(e) Deoxy/5′-eeekk kkeee 32 Me-DNA e = 2′-MOE, k = cEt

Example 55 Chimeric Oligonucleotides Comprising Modifications at theWing Regions Targeting HTT SNP

Additional gapmers are designed based on the gapmer selected fromstudies described in Tables 89 and 21 (ISIS 540107) and is marked withan asterisk (*). These gapmers are designed by introducing bicyclicmodified nucleosides at the 3′ or 5′ terminus and are tested to evaluateif the addition of bicyclic modified nucleosides at the wing regionsimproves the activity and selectivity in inhibition of mutant HTT SNP.

The gapmers comprise a 5-7-5 motif and are described in Table 81. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”, or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside, and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt).

TABLE 81 Modified oligonucleotides targeting HTT SNP wing SEQ ISISchemistry ID NO Sequence (5′ to 3′) Motif 5′ 3′ NO. 540107*A_(e)T_(e)A_(e)A_(e)A_(k)TTGTCATC_(k)A_(e)C_(e)C_(e)A_(e) 5-7-5 eeeekkeeee 32 (eeeek-d7-keeee) XXXX31A_(e)T_(e)A_(k)A_(k)A_(k)TTGTCATC_(k)A_(k)C_(k)C_(e)A_(e) 5-7-5 eekkkkkkee 32 (eekkk-d7-kkkee) XXXX32A_(e)T_(e)A_(e)A_(e)A_(k)TTGTCATC_(e)A_(e)C_(e)C_(e)A_(e) 5-7-5 eeeekeeeee 32 (eeeek-d7-eeeee) XXXX33A_(e)T_(e)A_(e)A_(k)A_(k)TTGTCATC_(e)A_(e)C_(e)C_(e)A_(e) 5-7-5 eeekkeeeee 32 (eeekk-d7-eeeee) XXXX34A_(e)T_(e)A_(k)A_(k)A_(k)TTGTCATC_(e)A_(e)C_(e)C_(e)A_(e) 5-7-5 eekkkeeeee 32 (eekkk-d7-eeeee) XXXX35A_(e)T_(e)A_(e)A_(e)A_(e)TTGTCATC_(k)A_(e)C_(e)C_(e)A_(e) 5-7-5 eeeeekeeee 32 (eeeee-d7-keeee) XXXX36A_(e)T_(e)A_(e)A_(e)A_(e)TTGTCATC_(k)A_(k)C_(e)C_(e)A_(e) 5-7-5 eeeeekkeee 32 (eeeee-d7-kkeee) XXXX37A_(e)T_(e)A_(e)A_(e)A_(e)TTGTCATC_(k)A_(k)C_(k)C_(e)A_(e) 5-7-5 eeeeekkkee 32 (eeeee-d7-kkkee) e = 2′-MOE; k = cEt; d =2′-deoxyribonucleoside

Example 56 Chimeric Oligonucleotides Comprising Wing and Central GapModifications Targeting HTT SNP

Additional gapmers are designed based on the parent gapmer, ISIS 460209,wherein the central gap region comprises nine 2′-deoxyribonucleosidesand is marked with an asterisk (*) in the table. These gapmers weredesigned by introducing modifications at the wings or the central gapregion and are created with a 3-9-3 motif.

The gapmers are described in Table 82. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytosinenucleobases throughout each gapmer are 5-methyl cytosines. Nucleosideswithout a subscript are β-D-2′-deoxyribonucleosides. Nucleosidesfollowed by a subscript “e”, or “k” are sugar modified nucleosides. Asubscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside,and a subscript “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g.cEt). ^(P)T indicates a 5-propyne thymidine nucleoside. ^(P)C indicatesa 5-propyne cytosine nucleoside. Underline nucleoside or the number inparentheses indicates the position on the oligonucleotides opposite tothe SNP position, as counted from the 5′-terminus.

TABLE 82 Modified oligonucleotides targeting HTT SNP wing SEQ ISISchemistry ID NO Sequence (5′ to 3′) Modification 5′ 3′ NO 460209* (8)T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) Deoxy gap ekk kke 10 (3-9-3)552103 (8) T_(e)A_(e)A_(e)ATTGTCATCA_(k)C_(k)C_(k) Deoxy gap eee kkk 10(3-9-3) 552104 (8) T_(k)A_(k)A_(k)ATTGTCATCA_(e)C_(e)C_(e) Deoxy gap kkkeee 10 (3-9-3) 552105 (8) T_(e)A_(k)A_(k)ATTG ^(P)T^(P)CATCA_(k)C_(k)C_(e) Deoxy/5- ekk kke 10 Propyne 552106 (8)T_(e)A_(k)A_(k)A^(P)T^(P)TG ^(P)T ^(P)CA^(P)T^(P)CA_(k)C_(k)C_(e)Deoxy/5- ekk kke 10 Propyne e = 2′-MOE; k = cEt

Example 57 Modified Oligonucleotides Comprising F-HNA Modification atthe Central Gap or Wing Region Targeting HTT SNP

A series of modified oligonucleotides were designed based on ISIS460209, wherein the central gap region contains nine2′-deoxyribonucleosides. These modified oligonucleotides were designedby incorporating one or more F-HNA(s) modification within the centralgap region or on the wing regions. The F-HNA containing oligonucleotideswere tested for their ability to selectively inhibit mutant (mut) HTTmRNA expression levels targeting rs7685686 while leaving the expressionof the wild-type (wt) intact. The activity and selectivity of themodified oligonucleotides were evaluated and compared to ISIS 460209.

The modified oligonucleotides and their motifs are described in Table83. The internucleoside linkages throughout each modifiedoligonucleotide are phosphorothioate linkages (P═S). Nucleosides withouta subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by asubscript “e” indicate 2′-O-methoxyethyl (MOE) modified nucleosides.Nucleosides followed by a subscript “k” indicate 6′-(S)—CH₃ bicyclicnucleosides (e.g. cEt). Nucleosides followed by a subscript “z” indicateF-HNA modified nucleosides. ^(m)C indicates a 5-methyl cytosinenucleoside. Underlined nucleoside indicates the position on theoligonucleotides opposite to the SNP position, which is position 8 ascounted from the 5′-terminus.

The gap-interrupted antisense oligonucleotides were tested in vitro.Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022cells at a density of 25,000 cells per well were transfected usingelectroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations ofmodified oligonucleotides. After a treatment period of approximately 16hours, RNA was isolated from the cells and mRNA levels were measured byquantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. The HTT mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN and the results arepresented in Table 84.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

The parent gapmer, 460209 is marked with an asterisk (*) in the tableand was included in the study as a benchmark oligonucleotide againstwhich the activity and selectivity of antisense oligonucleotidestargeting nucleotides overlapping the SNP position could be compared.

As illustrated in Table 84, oligonucleotides comprising F-HNAmodification(s) showed improvement in selectivity while maintainingactivity as compared to the parent gapmer, ISIS 460209.

TABLE 83 Gap-interrupted antisense oligonucleotides targeting HTT SNPWing SEQ ISIS Sequence Gap chemistry ID NO. (5′ to 3′) Motif chemistry5′ 3′ NO. 460209* T_(e)A_(k)A^(k)ATTGT 3-9-3 Full deoxy ekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 566266 T_(e)A_(k)A_(k)A_(z)TTGT3-9-3 or Deoxy/F- ekk or kke 10 ^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e)4-8-3 HNA ekkz 566267 T_(e)A_(k)A_(k)AT_(z)TGT 3-9-3 or Deoxy/F- ekk orkke 10 ^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 5-7-3 HNA ekkdz 566268T_(e)A_(k)A_(k)ATT_(z)GT 3-9-3 or Deoxy/F- ekk or kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 6-6-3 HNA ekkddz 566269T_(e)A_(k)A_(k)ATTG_(z) T 3-9-3 or Deoxy/F- ekk or kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 7-5-3 HNA ekkdddz 567369T_(e)A_(k)A_(k)A_(z)T_(z)TGT 3-9-3 or Deoxy/F- ekk or kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 5-7-3 HNA ekkzz e = 2′-MOE, k =cEt, d = 2′-β-deoxyribonucleoside, z = F-HNA

TABLE 84 Comparison of inhibition of HTT mRNA levels and selectivity ofgap-interrupted antisense oligonucleotides with ISIS 460209 targetingHTT SNP IC₅₀ (μM) Selectivity Gap Wing Chemistry ISIS NO Mut Wt (wt vsmut) Motif chemistry 5′ 3′ 460209* 0.28 3.1 11 3-9-3 Full deoxy ekk kke566266 0.20 >10 >50 3-9-3 or Deoxy/F- ekk or ekkz kke 4-8-3 HNA 5662670.90 >9.9 >11 3-9-3 or Deoxy/F- ekk or ekkdz kke 5-7-3 HNA 5662681.0 >10 >10 3-9-3 or Deoxy/F- ekk or ekkddz kke 6-6-3 HNA 5662691.7 >10.2 >6 3-9-3 or Deoxy/F- ekk or kke 7-5-3 HNA ekkdddz 5673690.82 >9.8 >12 3-9-3 or Deoxy/F- ekk or ekkzz kke 5-7-3 HNA e = 2′-MOE, k= cEt, d = 2′-β-deoxyribonucleoside, z = F-HNA

Example 58 Modified Oligonucleotides Comprising cEt Modification(s) atthe Central Gap Region Targeting HTT SNP

A series of modified oligonucleotides were designed in the same manneras described in Example 57.

These modified oligonucleotides were designed by replacing F-HNA(s) withcEt modification(s) in the central gap region while maintaining the wingconfiguration. The modified oligonucleotides were tested for theirability to selectively inhibit mutant (mut) HTT mRNA expression levelstargeting rs7685686 while leaving the expression of the wild-type (wt)intact. The activity and selectivity of the modified oligonucleotideswere evaluated and compared to ISIS 460209.

The modified oligonucleotides and their motifs are described in Table85. The internucleoside linkages throughout each modifiedoligonucleotide are phosphorothioate linkages (P═S). Nucleosides withouta subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by asubscript “e” indicate 2′-β-methoxyethyl (MOE) modified nucleosides.Nucleosides followed by a subscript “k” indicate 6′-(S)—CH₃ bicyclicnucleosides (e.g. cEt). ^(m)C indicates a 5-methyl cytosine nucleoside.Underlined nucleoside indicates the position on the oligonucleotidesopposite to the SNP position, which is position 8 as counted from the5′-terminus.

The gap-interrupted antisense oligonucleotides were tested in vitro.Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022cells at a density of 25,000 cells per well were transfected usingelectroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations ofmodified oligonucleotides. After a treatment period of approximately 16hours, RNA was isolated from the cells and mRNA levels were measured byquantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. The HTT mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN and the results arepresented below.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 86, some of the newly designed antisenseoligonucleotides (ISIS 575006, 575007, and 575008) showed improvement inpotency and/or selectivity in inhibiting mut HTT mRNA levels comparingto ISIS 460209.

TABLE 85 Gap-interrupted antisense oligonucleotides targeting HTT SNPWing SEQ ISIS Sequence Gap chemistry ID NO. (5′ to 3′) Motif chemistry5′ 3′ NO. 460209* T_(e)A_(k)A_(k)ATTGT 3-9-3 Full deoxy ekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 575006 T_(e)A_(k)A_(k)A_(k)TTGT4-8-3 Full deoxy ekkk kke 10 ^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e)575007 T_(e)A_(k)A_(k)AT_(k)TGT 3-9-3 or Full deoxy or ekk or kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 5-7-3 Deoxy/cEt ekkdk 575133T_(e)A_(k)A_(k)ATT_(k)GT 3-9-3 or Full deoxy or ekk or kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 6-6-3 Deoxy/cEt ekkddk 575134T_(e)A_(k)A_(k)ATTG_(k) T 3-9-3 or Full deoxy or ekk or kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 7-5-3 Deoxy/cEt ekkdddk 575008T_(e)A_(k)A_(k)A_(k)T_(k)TGT 5-7-3 Deoxy ekkkk kke 10 ^(m)CAT^(m)CA_(k)^(m)C_(k) ^(m)C_(e) e = 2′-MOE, k = cEt, d = 2′-β-deoxyribonucleoside

TABLE 86 Comparison of inhibition of HTT mRNA levels and selectivity ofgap-interrupted antisense oligonucleotides with ISIS 460209 targetingHTT SNP IC₅₀ (μM) Selectivity Gap Wing Chemistry ISIS NO Mut Wt (wt vsmut) Motif chemistry 5′ 3′ 460209* 0.28 3.1 11 3-9-3 Full deoxy ekk kke575006 0.27 3.8 14 4-8-3 Full deoxy ekkk kke 575007 0.67 >10.1 >15 3-9-3or Full deoxy or ekk or kke 5-7-3 Deoxy/cEt ekkdk 575133 3.0 >9 >3 3-9-3or Full deoxy or ekk or kke 6-6-3 Deoxy/cEt ekkddk 575134 2.6 >10.4 >43-9-3 or Full deoxy or ekk or kke 7-5-3 Deoxy/cEt ekkdddk 5750080.18 >9.9 >55 5-7-3 Full deoxy ekkkk kke e = 2′-MOE, k = cEt, d =2′-β-deoxyribonucleoside

Example 59 Modified Oligonucleotides Comprising F-HNA Modification atthe 3′-End of Central Gap Region Targeting HTT SNP

A series of modified oligonucleotides were designed based on ISIS460209, wherein the central gap region contains nine2′-deoxyribonucleosides. These modified oligonucleotides were designedby incorporating one F-HNA modification at the 3′-end of the central gapregion. The F-HNA containing oligonucleotides were tested for theirability to selectively inhibit mutant (mut) HTT mRNA expression levelstargeting HTT SNP while leaving the expression of the wild-type (wt)intact. The activity and selectivity of the modified oligonucleotideswere evaluated and compared to ISIS 460209.

The modified oligonucleotides and their motifs are described in Table87. The internucleoside linkages throughout each modifiedoligonucleotide are phosphorothioate linkages (P═S). Nucleosides withouta subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by asubscript “e” indicate 2′-β-methoxyethyl (MOE) modified nucleosides.Nucleosides followed by a subscript “k” indicate 6′-(S)—CH₃ bicyclicnucleosides (e.g. cEt). Nucleosides followed by a subscript “z” indicateF-HNA modified nucleosides. ^(m)C indicates a 5-methyl cytosinenucleoside. Underlined nucleoside indicates the position on theoligonucleotides opposite to the SNP position, which is position 8 ascounted from the 5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modifiedoligonucleotides. After a treatment period of approximately 16 hours,RNA was isolated from the cells and mRNA levels were measured byquantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. The HTT mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN and the results arepresented in Table 88.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 88, a couple of the newly designed antisenseoligonucleotides (ISIS 575833 and 575834) showed improvement inselectivity while maintaining potency as compared to ISIS 460209. ISIS575836 showed an increase in potency without improvement in selectivitywhile ISIS 575835 showed comparable selectivity without improvement inpotency.

TABLE 87 Modified oligonucleotides targeting HTT SNP Gap Wing SEQ ISISSequence  chem- chemistry ID NO. (5′ to 3′) Motif istry 5′ 3′ NO.460209* T_(e)A_(k)A_(k)ATTGT 3-9-3 Full  ekk kke 10 ^(m)CAT^(m)CA_(k)^(m)C_(k) ^(m)C_(e) deoxy 575833 T_(e)A_(k)A_(k)ATTGT 3-9-3  Deoxy/ ekkkke or 10 ^(m)C_(z)AT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) or F-HNA zdddkke3-5-7 575834 T_(e)A_(k)A_(k)ATTGT 3-9-3  Deoxy/ ekk kke or 10^(m)CA_(z)T^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) or F-HNA zddkke 3-6-6 575835T_(e)A_(k)A_(k)ATTGT 3-9-3  Deoxy/ ekk kke or 10 ^(m)CAT_(z) ^(m)CA_(k)^(m)C_(k) ^(m)C_(e) or F-HNA zdkke 3-7-5 575836 T_(e)A_(k)A_(k)ATTGT3-9-3  Deoxy/ ekk  kke or 10 ^(m)CAT^(m)C_(z)A_(k) ^(m)C_(k) ^(m)C_(e)or F-HNA zkke 3-8-4 e = 2′-MOE, k = cEt, d = 2′-β-deoxyribonucleoside, z= F-HNA

TABLE 88 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeting HTT SNP IC₅₀ (μM)Selectivity Wing Chemistry ISIS NO Mut Wt (wt vs mut) Motif Gapchemistry 5′ 3′ 460209* 0.28 3.1 11 3-9-3 Full deoxy ekk kke 575833 0.224.2 19 3-9-3 or Deoxy/F-HNA ekk kke or 3-5-7 zdddkke 575834 0.30 6.3 213-9-3 or Deoxy/F-HNA ekk kke or 3-6-6 zddkke 575835 0.89 9.8 11 3-9-3 orDeoxy/F-HNA ekk kke or 3-7-5 zdkke 575836 0.09 0.4 4.6 3-9-3 orDeoxy/F-HNA ekk kke or zkke 3-8-4 e = 2′-MOE, k = cEt, d =2′-β-deoxyribonucleoside, z = F-HNA

Example 60 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin(HTT) Single Nucleotide Polymorphism (SNP)

Additional chimeric antisense oligonucleotides were designed based onISIS 460209 and ISIS 540094 wherein the central gap region contains nine2′-deoxynucleosides. These gapmers were designed with the central gapregion shortened by introducing cEt modifications to the wing regions,or interrupted by introducing cEt modifications at the 3′-end of thecentral gap region. The modified oligonucleotides were tested for theirability to selectively inhibit mutant (mut) HTT mRNA expression levelstargeting HTT SNP while leaving the expression of the wild-type (wt)intact. The activity and selectivity of the modified oligonucleotideswere evaluated and compared to ISIS 460209 and 540094.

The gapmers and their motifs are described in Table 89. Theinternucleoside linkages throughout each modified oligonucleotide arephosphorothioate linkages (P═S). Nucleosides without a subscript are(3-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosidesfollowed by a subscript “k” indicate 6′-(S)—CH₃ bicyclic nucleosides(e.g. cEt). ^(m)C indicates a 5-methyl cytosine nucleoside. Underlinednucleoside indicates the position on the oligonucleotides opposite tothe SNP position, which is position 4 or 8 as counted from the5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modifiedoligonucleotides. After a treatment period of approximately 16 hours,RNA was isolated from the cells and mRNA levels were measured byquantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. The HTT mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN and the results arepresented in Table 90.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 90, the newly designed antisenseoligonucleotides (ISIS 575003) showed improvement in selectivity whilemaintaining potency as compared to ISIS 460209.

TABLE 89 Short-gap antisense oligonucleotides  targeting HTT SNP WingSEQ ISIS Sequence Gap chemistry ID NO. (5′ to 3′) Motif chemistry 5′ 3′NO. 460209* T_(e)A_(k)A_(k)ATTGT 3-9-3 Full  ekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) deoxy 540094* T_(e)T_(k)GT^(m)CAT^(m)CA 2-9-4 Full  ek kkke 67 ^(m)C^(m)CA_(k)G_(k)A_(k)A_(e)deoxy 575003 T_(e)T_(k)GT ^(m)CAT^(m)CA 2-8-5 Full  ek kkkke 67^(m)C^(m)C_(k)A_(k)G_(k)A_(k)A_(e) deoxy 575004 T_(e)T_(k)GT^(m)CAT^(m)CA 2-9-4  Full   ek kkke  67 ^(m)C_(k)^(m)CA_(k)G_(k)A_(k)A_(e) or deoxy or 2-7-6 or kdkkke Deoxy/cEt 575005T_(e)T_(k)GT ^(m)CAT^(m)CA 2-7-6 Full  ek kkkkke 67 ^(m)C_(k)^(m)C_(k)A_(k)G_(k)A_(k)A_(e) deoxy e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

TABLE 90 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeting HTT SNP IC₅₀ (μM)Selectivity Wing Chemistry ISIS NO Mut Wt (wt vs mut) Motif Gapchemistry 5′ 3′ 460209* 0.34 3.3 9.7 3-9-3 Full deoxy ekk kke 540094*0.17 2.4 14 2-9-4 Full deoxy ek kkke 575003 0.40 10 25 2-8-5 Full deoxyek kkkke 575004 1.2 >9.6 >8 2-9-4 or Full deoxy or ek kkke or 2-7-6Deoxy/cEt kdkkke 575005 >10 >100 >10 2-7-6 Full deoxy ek kkkkke e =2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 61 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin(HTT) Single Nucleotide Polymorphism (SNP)

Additional chimeric antisense oligonucleotides were designed based on15-mer, ISIS 460209 and 17-mer, ISIS 476333 wherein the central gapregion contains nine 2′-deoxynucleosides. These gapmers were designedwith the central gap region shortened at the 5′-end of the central gapregion. The gapmers were tested for their ability to selectively inhibitmutant (mut) HTT mRNA expression levels targeting HTT SNP while leavingthe expression of the wild-type (wt) intact. The activity andselectivity of the gapmers were evaluated and compared to ISIS 460209and ISIS 476333.

The gapmers and their motifs are described in Table 91. Theinternucleoside linkages throughout each modified oligonucleotide arephosphorothioate linkages (P═S). Nucleosides without a subscript are(3-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosidesfollowed by a subscript “k” indicate 6′-(S)—CH₃ bicyclic nucleosides(e.g. cEt). ^(m)C indicates a 5-methyl cytosine nucleoside. Underlinednucleoside indicates the position on the oligonucleotides opposite tothe SNP position, which is position 8 or 9 as counted from the5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modifiedoligonucleotides. After a treatment period of approximately 16 hours,RNA was isolated from the cells and mRNA levels were measured byquantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. The HTT mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN and the results arepresented in Table 92.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 92, a couple of the newly designed antisenseoligonucleotides (ISIS 571036 and 571037) showed improvement in potencyand selectivity in inhibiting mut HTT mRNA levels as compared to ISIS460209 and 476333.

TABLE 91 Short-gap antisense oligonucleotides  targeting HTT SNP GapWing SEQ ISIS Sequence chem- chemistry ID NO. (5′ to 3′) Motif istry 5′3′ NO. 460209* T_(e)A_(k)A_(k)ATTGT 3-9-3 Full  ekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) deoxy 476333*A_(e)T_(k)A_(e)A_(k)ATTGT 4-9-4 Full  ekek keke 32 ^(m)CAT^(m)CA_(k)^(m)C_(e) ^(m)C_(k)A_(e) deoxy 571036 A_(e)T_(k)A_(e)A_(k)A_(e)T_(k)TGT6-7-4 Full  ekekek keke 32 ^(m)CAT^(m)CA_(k) ^(m)C_(e) ^(m)C_(k)A_(e)deoxy 571037 A_(e)T_(e)A_(e)A_(e)A_(k)T_(k)TGT 6-7-4 Full  eeeekk keke32 ^(m)CAT^(m)CA_(k) ^(m)C_(e) ^(m)C_(k)A_(e) deoxy 571038A_(e)T_(k)A_(e)A_(k)A_(e)T_(e)TGT 6-7-4 Full  ekekee keke 32^(m)CAT^(m)CA_(k) ^(m)C_(e) ^(m)C_(k)A_(e) deoxy e = 2′-MOE, k = cEt, d= 2′-deoxyribonucleoside

TABLE 92 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeting HTT SNP SelectivityWing IC₅₀ (μM) (wt vs Gap Chemistry ISIS NO Mut Wt mut) Motif chemistry5′ 3′ 460209* 0.34 3.3 9.7 3-9-3 Full deoxy ekk kke 476333* 0.32 1.5 4.74-9-4 Full deoxy ekek keke 571036 0.17 >10.0 >59 6-7-4 Full deoxy ekekekkeke 571037 0.11 >9.9 >90 6-7-4 Full deoxy eeeekk keke 5710381.5 >10.5 >7 6-7-4 Full deoxy ekekee keke e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

Example 62 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin(HTT) Single Nucleotide Polymorphism (SNP)

Additional chimeric antisense oligonucleotides were designed based on15-mer, ISIS 460209 wherein the central gap region contains nine2′-deoxynucleosides. These gapmers were designed by having the centralgap region shortened to seven 2′-deoxynucleosides. The gapmers weretested for their ability to selectively inhibit mutant (mut) HTT mRNAexpression levels targeting HTT SNP while leaving the expression of thewild-type (wt) intact. The activity and selectivity of the gapmers wereevaluated and compared to ISIS 460209.

The gapmers and their motifs are described in Table 93. Theinternucleoside linkages throughout each modified oligonucleotide arephosphorothioate linkages (P═S). Nucleosides without a subscript are13-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosidesfollowed by a subscript “k” indicate 6′-(S)—CH₃ bicyclic nucleosides(e.g. cEt). ^(m)C indicates a 5-methyl cytosine nucleoside. Underlinednucleoside indicates the position on the oligonucleotides opposite tothe SNP position, which is position 8 or 9 as counted from the5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modifiedoligonucleotides. After a treatment period of approximately 16 hours,RNA was isolated from the cells and mRNA levels were measured byquantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. The HTT mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN and the results arepresented in Table 94.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 94, each of the newly designed antisenseoligonucleotides (ISIS 540108 and 571069) showed improvement in potencyand/or selectivity in inhibiting mut HTT mRNA levels as compared to ISIS460209.

TABLE 93 Short-gap antisense oligonucleotides  targeting HTT SNP GapWing SEQ ISIS Sequence Mo- chem- chemistry ID NO. (5′ to 3′) tif istry5′ 3′ NO. 460209 T_(e)A_(k)A_(k)ATTGT 3-9- Full  ekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 3 deoxy 540108A_(e)T_(e)A_(e)A_(k)A_(k)TTGT 5-7- Full  eeekk kkeee 32^(m)CAT^(m)C_(k)A_(k) ^(m)C_(e) ^(m)C_(e)A_(e) 5 deoxy 571069A_(e)T_(e)A_(e)A_(e)A_(k)T_(k)TGT 6-7- Full  eeeekk kkee 32^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e)A_(e) 4 deoxy 571173A_(e)T_(e)A_(k)A_(k)ATTGT 4-7- Full  eekk kkeeee 32 ^(m)CAT_(k)^(m)C_(k)A_(e) ^(m)C_(e) ^(m)C_(e)A_(e) 6 deoxy 572773T_(e)A_(e)A_(k)A_(k)TTGT 4-7- Full  eekk kkee 10 ^(m)CAT^(m)C_(k)A_(k)^(m)C_(e) ^(m)C_(e) 4 deoxy e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

TABLE 94 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeting HTT SNP SelectivityISIS IC₅₀ (μM) (wt vs Gap Wing Chemistry NO Mut Wt mut) Motif chemistry5′ 3′ 460209 0.34 3.3 9.7 3-9-3 Full deoxy ekk kke 540108 0.20 >10 >505-7-5 Full deoxy eeekk kkeee 571069 0.29 >9.9 >34 6-7-4 Full deoxyeeeekk kkee 571173 1.0 >10 >10 4-7-6 Full deoxy eekk kkeeee 5727730.71 >7.8 11 4-7-4 Full deoxy eekk kkee e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

Example 63 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin(HTT) Single Nucleotide Polymorphism (SNP)

Additional chimeric antisense oligonucleotides were designed based on15-mer, ISIS 460209 and 17-mer, ISIS 540108 wherein the central gapregion contains nine and seven 2′-deoxynucleosides, respectively. Thesegapmers were designed by introducing one or more cEt modification(s) atthe 5′-end of the central gap region. The gapmers were tested for theirability to selectively inhibit mutant (mut) HTT mRNA expression levelstargeting HTT SNP while leaving the expression of the wild-type (wt)intact. The activity and selectivity of the gapmers were evaluated andcompared to ISIS 460209 and ISIS 540108.

The gapmers and their motifs are described in Table 95. Theinternucleoside linkages throughout each modified oligonucleotide arephosphorothioate linkages (P═S). Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosidesfollowed by a subscript “k” indicate 6′-(S)—CH₃ bicyclic nucleosides(e.g. cEt). ^(m)C indicates a 5-methyl cytosine nucleoside. Underlinednucleoside indicates the position on the oligonucleotides opposite tothe SNP position, which is position 8 or 9 as counted from the5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modifiedoligonucleotides. After a treatment period of approximately 16 hours,RNA was isolated from the cells and mRNA levels were measured byquantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. The HTT mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN and the results arepresented in Table 96.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 96, most of the newly designed oligonucleotidesshowed improvement in selectivity while maintaining potency as comparedto 460209.

TABLE 95 Short-gap antisense oligonucleotides  targeting HTT SNP GapWing SEQ ISIS Sequence chem- chemistry ID NO. (5′ to 3′) Motif istry 5′3′ NO. 460209 T_(e)A_(k)A_(k)ATTGT 3-9-3 Full  ekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) deoxy 540108A_(e)T_(e)A_(e)A_(k)A_(k)TTGT 5-7-5 Full  eeekk kkeee 32^(m)CAT^(m)C_(k)A_(k) ^(m)C_(e) ^(m)C_(e)A_(e) deoxy 556872A_(e)T_(e)A_(e)A_(e)A_(k)TTGT 5-7-5 Full  eeeek eeeee 32^(m)CAT^(m)C_(e)A_(e) ^(m)C_(e) ^(m)C_(e)A_(e) deoxy 556873A_(e)T_(e)A_(e)A_(k)A_(k)TTGT 5-7-5 Full  eeekk eeeee 32^(m)CAT^(m)C_(e)A_(e) ^(m)C_(e) ^(m)C_(e)A_(e) deoxy 556874A_(e)T_(e)A_(k)A_(k)A_(k)TTGT 5-7-5 Full  eekkk eeeee 32^(m)CAT^(m)C_(e)A_(e) ^(m)C_(e) ^(m)C_(e)A_(e) deoxy 568877A_(e)T_(k)A_(k)A_(k)A_(k)TTGT 5-7-5 Full  ekkkk eeeee 32^(m)CAT^(m)C_(e)A_(e) ^(m)C_(e) ^(m)C_(e)A_(e) deoxy 568878A_(k)T_(k)A_(k)A_(k)A_(k)TTGT 5-7-5 Full  kkkkk eeeee 32^(m)CAT^(m)C_(e)A_(e) ^(m)C_(e) ^(m)C_(e)A_(e) deoxy e = 2′-MOE, k =cEt, d = 2′-deoxyribonucleoside

TABLE 96 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeting HTT SNP Wing IC₅₀(μM) Selectivity Gap Chemistry ISIS NO Mut Wt (wt vs mut) Motifchemistry 5′ 3′ 460209 0.45 2.3 5.1 3-9-3 Full deoxy ekk kke 540108 0.259.5 38 5-7-5 Full deoxy eeekk kkeee 556872 1.0 9.9 9.9 5-7-5 Full deoxyeeeek eeeee 556873 0.67 3.4 5.1 5-7-5 Full deoxy eeekk eeeee 556874 0.381.9 5.0 5-7-5 Full deoxy eekkk eeeee 568877 0.44 6.2 14 5-7-5 Full deoxyekkkk eeeee 568878 0.41 8.6 21 5-7-5 Full deoxy kkkkk eeeee e = 2′-MOE,k = cEt, d = 2′-deoxyribonucleoside

Example 64 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin(HTT) Single Nucleotide Polymorphism (SNP)

Additional chimeric antisense oligonucleotides were designed based on15-mer, ISIS 460209 and 17-mer, ISIS 540108 wherein the central gapregion contains nine and seven 2′-deoxynucleosides, respectively. Thesegapmers were designed by introducing one or more cEt modification(s) atthe 3′-end of the central gap region. The gapmers were tested for theirability to selectively inhibit mutant (mut) HTT mRNA expression levelstargeting HTT SNP while leaving the expression of the wild-type (wt)intact. The activity and selectivity of the gapmers were evaluated andcompared to ISIS 460209 and ISIS 540108.

The gapmers and their motifs are described in Table 97. Theinternucleoside linkages throughout each modified oligonucleotide arephosphorothioate linkages (P═S). Nucleosides without a subscript are(3-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosidesfollowed by a subscript “k” indicate 6′-(S)—CH₃ bicyclic nucleosides(e.g. cEt). ^(m)C indicates a 5-methyl cytosine nucleoside. Underlinednucleoside indicates the position on the oligonucleotides opposite tothe SNP position, which is position 8 or 9 as counted from the5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modifiedoligonucleotides. After a treatment period of approximately 16 hours,RNA was isolated from the cells and mRNA levels were measured byquantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. The HTT mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN and the results arepresented in Table 98.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 98, each of the newly designed oligonucleotidesshowed improvement in selective inhibition of mutant HTT mRNA levelscompared to ISIS 460209. Comparable potency was observed for ISIS 568879and 568880 while a slight loss in potency was observed for ISIS 556875,556876 and 556877.

TABLE 97 Short-gap antisense oligonucleotides  targeting HTT SNP GapWing SEQ ISIS Sequence chem- chemistry ID NO. (5′ to 3′) Motif istry 5′3′ NO. 460209 T_(e)A_(k)A_(k)ATTGT 3-9-3 Full  ekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) deoxy 540108A_(e)T_(e)A_(e)A_(k)A_(k)TTGT 5-7-5 Full  eeekk kkeee 32^(m)CAT^(m)C_(k)A_(k) ^(m)C_(e) ^(m)C_(e)A_(e) deoxy 556875A_(e)T_(e)A_(e)A_(e)A_(e)TTGT 5-7-5 Full  eeeee keeee 32^(m)CAT^(m)C_(k)A_(e) ^(m)C_(e) ^(m)C_(e)A_(e) deoxy 556876A_(e)T_(e)A_(e)A_(e)A_(e)TTGT 5-7-5 Full  eeeee kkeee 32^(m)CAT^(m)C_(k)A_(k) ^(m)C_(e) ^(m)C_(e)A_(e) deoxy 556877A_(e)T_(e)A_(e)A_(e)A_(e)TTGT 5-7-5 Full  eeeee kkkee 32^(m)CAT^(m)C_(k)A_(k) ^(m)C_(k) ^(m)C_(e)A_(e) deoxy 568879A_(e)T_(e)A_(e)A_(e)A_(e)TTGT 5-7-5 Full  eeeee kkkke 32^(m)CAT^(m)C_(k)A_(k) ^(m)C_(k) ^(m)C_(k)A_(e) deoxy 568880A_(e)T_(e)A_(e)A_(e)A_(e)TTGT 5-7-5 Full  eeeee kkkkk 32^(m)CAT^(m)C_(k)A_(k) ^(m)C_(k) ^(m)C_(k)A_(k) deoxy e = 2′-MOE, k =cEt, d = 2′-deoxyribonucleoside

TABLE 98 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeting HTT SNP SelectivityWing IC₅₀ (μM) (wt vs Gap Chemistry ISIS NO Mut Wt mut) Motif chemistry5′ 3′ 460209 0.45 2.3 5.1 3-9-3 Full deoxy ekk kke 540108 0.25 9.5 385-7-5 Full deoxy eeekk kkeee 556875 1.9 >9.5 >5 5-7-5 Full deoxy eeeeekeeee 556876 0.99 >9.9 >10 5-7-5 Full deoxy eeeee kkeee 5568771.0 >10 >10 5-7-5 Full deoxy eeeee kkkee 568879 0.44 >10.1 >23 5-7-5Full deoxy eeeee kkkke 568880 0.59 >10 >17 5-7-5 Full deoxy eeeee kkkkke = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 65 Modified Oligonucleotides Targeting Huntingtin (HTT) SingleNucleotide Polymorphism (SNP)

A series of modified oligonucleotides were designed based on the parentgapmer, ISIS 460209 wherein the central gap region contains nine2′-deoxyribonucleosides. These modified oligonucleotides were designedby introducing various chemical modifications in the central gap regionand were tested for their ability to selectively inhibit mutant (mut)HTT mRNA expression levels targeting SNP while leaving the expression ofthe wild-type (wt) intact. The activity and selectivity of the modifiedoligonucleotides were evaluated and compared to the parent gapmer, ISIS460209.

The modified oligonucleotides were created with a 3-9-3 motif and aredescribed in Table 99. The internucleoside linkages throughout eachgapmer are phosphorothioate (P═S) linkages, except for theinternucleoside linkage having a subscript “p” which indicates a methylphosphonate internucleoside linkage (—O—P(CH₃)(═O)—O—). Nucleosideswithout a subscript are β-D-2′-deoxyribonucleosides. Nucleosidesfollowed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modifiednucleoside. Nucleosides followed by a subscript “k” indicates a6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt). ^(m)C indicates a 5-methylcytosine nucleoside. ^(x)T indicates a 2-thio-thymidine nucleoside.Underlined nucleoside indicates the position on the oligonucleotidesopposite to the SNP position, which is position 8 as counted from the5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used (from Coriell Institute). CulturedGM04022 cells at a density of 25,000 cells per well were transfectedusing electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrationsof modified oligonucleotides. After a treatment period of approximately24 hours, cells were washed with DPBS buffer and lysed. RNA wasextracted using Qiagen RNeasy purification and mRNA levels were measuredby quantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. RT-PCR method in short; A mixture was madeusing 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uLwater and 40.4 uL RT MIX. To each well was added 15 uL of this mixtureand 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels weremeasured simultaneously by using two different fluorophores, FAM formutant allele and VIC for wild-type allele. The HTT mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN andthe results are presented in Table 100.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 100, improvement in selectivity with a slightdecrease in potency was observed for the newly designed oligonucleotidesas compared to ISIS 460209.

TABLE 99 Short-gap antisense oligonucleotides  targeting HTT SNP WingSEQ ISIS Sequence  Gap chemistry ID NO. (5′ to 3′) chemistry 5′ 3′ NO.460209 T_(e)A_(k)A_(k)ATTGT Full deoxy ekk kke 10 ^(m)CAT^(m)CA_(k)^(m)C_(k) ^(m)C_(e) 556845 T_(e)A_(k)A_(k)A^(x)TTGT Deoxy/2-Thio ekk kke10 ^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 556847T_(e)A_(k)A_(k)A^(x)T^(x)TGT Deoxy/2-Thio ekk kke 10 ^(m)CAT^(m)CA_(k)^(m)C_(k) ^(m)C_(e) 558257 T_(e)A_(k)A_(k)ATT_(p)GT Deoxy/Methyl ekk kke10 ^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C Phosphonate 571125T_(e)A_(k)A_(k)A^(x)TT_(p)GT Deoxy/2-Thio/ ekk kke 10 ^(m)CAT^(m)CA_(k)^(m)C_(k) ^(m)C_(e) Methyl Phosphonate e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

TABLE 100 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeting HTT SNP Wing IC₅₀(μM) Selectivity Chemistry ISIS NO Mut Wt (wt vs mut) Gap chemistry 5′3′ 460209 0.56 3.8 6.8 Full deoxy ekk kke 556845 0.98 >9.8 >10Deoxy/2-Thio ekk kke 556847 1.3 >10.4 >8 Deoxy/2-Thio ekk kke 5582571.7 >10.2 >6 Deoxy/Methyl ekk kke Phosphonate 571125 1.8 >10.8 >6Deoxy/2- ekk kke Thio/Methyl Phosphonate e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

Example 66 Modified Oligonucleotides Comprising Chemical Modificationsin the Central Gap Region Targeting Huntingtin (HTT) Single NucleotidePolymorphism (SNP)

Additional chimeric antisense oligonucleotides were designed in the samemanner as the antisense oligonucleotides described in Example 65. Thesegapmers were designed by introducing various modifications in thecentral gap region and were tested for their ability to selectivelyinhibit mutant (mut) HTT mRNA expression levels targeting SNP whileleaving the expression of the wild-type (wt) intact. The activity andselectivity of the modified oligonucleotides were evaluated and comparedto the parent gapmer, ISIS 460209.

The modified oligonucleotides and their motifs are described in Table101. The internucleoside linkages throughout each gapmer arephosphorothioate (P═S) linkages, except for the internucleoside linkagehaving a subscript “p” which indicates a methyl phosphonateinternucleoside linkage (—O—P(CH₃)(═O)—O—). Nucleosides without asubscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by asubscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside.Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH₃ bicyclicnucleoside (e.g. cEt). ^(m)C indicates a 5-methyl cytosine nucleoside.^(x)T indicates a 2-thio-thymidine nucleoside. Underlined nucleosideindicates the position on the oligonucleotides opposite to the SNPposition, which is position 8 as counted from the 5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used (from Coriell Institute). CulturedGM04022 cells at a density of 25,000 cells per well were transfectedusing electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrationsof modified oligonucleotides. After a treatment period of approximately24 hours, cells were washed with DPBS buffer and lysed. RNA wasextracted using Qiagen RNeasy purification and mRNA levels were measuredby quantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. RT-PCR method in short; A mixture was madeusing 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uLwater and 40.4 uL RT MIX. To each well was added 15 uL of this mixtureand 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels weremeasured simultaneously by using two different fluorophores, FAM formutant allele and VIC for wild-type allele. The HTT mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN andthe results are presented in Table 102.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 102, some of the newly designed oligonucleotidesshowed improvement in selectivity while maintaining potency as comparedto 460209.

TABLE 101 Short-gap antisense oligonucleotides targeting HTT SNP WingSEQ ISIS Sequence Gap chemistry ID NO. (5′ to 3′) Motif chemistry 5′ 3′NO. 460209 T_(e)A_(k)A_(k)ATTGT 3-9-3 Full deoxy ekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 551429T_(e)A_(e)A_(e)A_(k)T_(k)TGT 5-7-3 Full deoxy eeekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 571122 T_(e)A_(e)A_(e)A_(k)^(x)TTGT 4-8-3 Deoxy/2-Thio eeek kke 10 ^(m)CAT^(m)CA_(k) ^(m)C_(k)^(m)C_(e) 571123 T_(e)A_(e)A_(e)A_(k)T_(k)T_(p)GT 5-7-3 Deoxy/Methyleeekk kke 10 ^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) Phosphonate 571124T_(e)A_(e)A_(e)A_(k) ^(x)TT_(p)GT 4-8-3 Deoxy/2- eeek kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) Thio/Methyl Phosphonate 579854T_(e)A_(e)A_(e)A_(k)TT_(p)GT 4-8-3 Deoxy/Methyl eeek kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) Phosphonate 566282T_(e)A_(k)A_(k)A_(dx)T_(dx)T_(d)G_(d)T_(d) ^(m)C_(d) 3-9-3 Deoxy/Methylekk kke 10 A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) Phosphonate e =2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

TABLE 102 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeting HTT SNP SelectivityWing ISIS IC₅₀ (μM) (wt vs Chemistry NO Mut Wt mut) Motif Gap chemistry5′ 3′ 460209 0.56 3.8 6.8 3-9-3 Full deoxy ekk kke 551429 0.50 >10 >205-7-3 Full deoxy eeekk kke 571122 1.8 >10.8 >6 4-8-3 Deoxy/2-Thio eeekkke 571123 0.96 >9.6 >10 5-7-3 Deoxy/Methyl eeekk kke Phosphonate 5711242.3 >9.2 >4 4-8-3 Deoxy/2- eeek kke Thio/Methyl Phosphonate 5798540.63 >10.1 >16 4-8-3 Deoxy/Methyl eeek kke Phosphonate 566282 0.51 6.312.4 3-9-3 Deoxy/Methyl ekk kke Phosphonate e = 2′-MOE, k = cEt

Example 67 Modified Oligonucleotides Comprising Chemical Modificationsin the Central Gap Region Targeting Huntingtin (HTT) Single NucleotidePolymorphism (SNP)

Additional chimeric antisense oligonucleotides were designed in the samemanner as the antisense oligonucleotides described in Example 65. Thesegapmers were designed by introducing various modifications in thecentral gap region and were tested for their ability to selectivelyinhibit mutant (mut) HTT mRNA expression levels targeting SNP whileleaving the expression of the wild-type (wt) intact. The activity andselectivity of the modified oligonucleotides were evaluated and comparedto the parent gapmer, ISIS 460209.

The modified oligonucleotides and their motifs are described in Table103. The internucleoside linkages throughout each gapmer arephosphorothioate (P═S) linkages, except for the internucleoside linkagehaving a subscript “p” which indicates a methyl phosphonateinternucleoside linkage (—O—P(CH₃)(═O)—O—). Nucleosides without asubscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by asubscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside.Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH₃ bicyclicnucleoside (e.g. cEt). ^(m)C indicates a 5-methyl cytosine nucleoside.^(x)T indicates a 2-thio-thymidine nucleoside. Underlined nucleosideindicates the position on the oligonucleotides opposite to the SNPposition, which is position 8 or 9 as counted from the 5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used (from Coriell Institute). CulturedGM04022 cells at a density of 25,000 cells per well were transfectedusing electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrationsof modified oligonucleotides. After a treatment period of approximately24 hours, cells were washed with DPBS buffer and lysed. RNA wasextracted using Qiagen RNeasy purification and mRNA levels were measuredby quantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. RT-PCR method in short; A mixture was madeusing 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uLwater and 40.4 uL RT MIX. To each well was added 15 uL of this mixtureand 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels weremeasured simultaneously by using two different fluorophores, FAM formutant allele and VIC for wild-type allele. The HTT mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN andthe results are presented in Table 104.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 104, all but one of the newly designedoligonucleotides showed improvement in selectivity while maintainingpotency as compared to ISIS 460209.

TABLE 103 Short-gap antisense oligonucleotides targeting  HTT SNP WingSEQ ISIS Sequence Gap chemistry ID NO. (5′ to 3′) Motif chemistry 5′ 3′NO. 460209 T_(e)A_(k)A_(k)ATTGT 3-9-3 Full  ekk kke 10 ^(m)CAT^(m)CA_(k)^(m)C_(k) ^(m)C_(e) deoxy 476333 A_(e)T_(k)A_(e)A_(k)ATTGT 4-9-4 Full ekek keke 32 ^(m)CAT^(m)CA_(k) ^(m)C_(e) ^(m)C_(k)A_(e) deoxy 571039A_(e)T_(k)A_(e)A_(k)A^(x)TTGT 4-9-4 Deoxy/ ekek keke 32^(m)CAT^(m)CA_(k) ^(m)C_(e) ^(m)C_(k)A_(e) 2-Thio 571171A_(e)T_(k)A_(e)A_(k)ATT_(p)GT 4-9-4 Deoxy/ ekek keke 32^(m)CAT^(m)CA_(k) ^(m)C_(e) ^(m)C_(k)A_(e) Methyl Phospho- nate 571041A_(e)T_(k)A_(e)A_(k)A^(x)TT_(p)GT 4-9-4 Deoxy/2- ekek keke 32^(m)CAT^(m)CA_(k) ^(m)C_(e) ^(m)C_(k)A_(e) Thio/ Methyl Phospho- nate e= 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

TABLE 104 Comparison of inhibition of HTT mRNA levels and selectivity ofmodified oligonucleotides with ISIS 460209 targeting HTT SNP ISIS IC₅₀(μM) Selectivity Gap Wing Chemistry NO Mut Wt (wt vs mut) chemistry 5′3′ 460209 0.56 3.8 6.8 Full deoxy ekk kke 476333 0.56 3.4 6.1 Full deoxyekek keke 571039 0.34 >9.9 >29 Deoxy/2-Thio ekek keke 5711710.54 >10.3 >19 Deoxy/Methyl ekek keke Phosphonate 571041 0.75 >9.8 >13Deoxy/2- ekek keke Thio/Methyl Phosphonate e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

Example 68 Selectivity in Inhibition of HTT mRNA Levels Targeting SNP byGap-Interrupted Modified Oligonucleotides

Additional modified oligonucleotides were designed based on the parentgapmer, ISIS 460209 wherein the central gap region contains nine2′-deoxyribonucleosides. These modified oligonucleotides were designedby introducing one or more modified nucleobase(s) in the central gapregion and were tested for their ability to selectively inhibit mutant(mut) HTT mRNA expression levels targeting SNP while leaving theexpression of the wild-type (wt) intact. The activity and selectivity ofthe modified oligonucleotides were evaluated and compared to ISIS460209.

The modified oligonucleotides were created with a 3-9-3 motif and aredescribed in Table 105. The internucleoside linkages throughout eachgapmer are phosphorothioate (P═S) linkages. Nucleosides without asubscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by asubscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside.Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH₃ bicyclicnucleoside (e.g. cEt). ^(m)C indicates a 5-methyl cytosine nucleoside.^(x)T indicates a 2-thio-thymidine nucleoside. Underlined nucleosideindicates the position on the oligonucleotides opposite to the SNPposition, which is position 8 as counted from the 5′-terminus.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used (from Coriell Institute). CulturedGM04022 cells at a density of 25,000 cells per well were transfectedusing electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrationsof modified oligonucleotides. After a treatment period of approximately24 hours, cells were washed with DPBS buffer and lysed. RNA wasextracted using Qiagen RNeasy purification and mRNA levels were measuredby quantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. RT-PCR method in short; A mixture was madeusing 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uLwater and 40.4 uL RT MIX. To each well was added 15 uL of this mixtureand 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels weremeasured simultaneously by using two different fluorophores, FAM formutant allele and VIC for wild-type allele. The HTT mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN.

The IC₅₀ and selectivity were calculated using methods previouslydescribed in Example 41. The IC₅₀ at which each oligonucleotide inhibitsthe mutant HTT mRNA expression is denoted as ‘mut IC₅₀’. The IC₅₀ atwhich each oligonucleotide inhibits the wild-type HTT mRNA expression isdenoted as ‘wt IC₅₀’. Selectivity was calculated by dividing the IC₅₀for inhibition of the wild-type HTT versus the IC₅₀ for inhibitingexpression of the mutant HTT mRNA.

As illustrated in Table 106, ISIS 556845 showed improvement inselectivity and potency as compared to ISIS 460209. ISIS 556847 showedimprovement in selectivity with comparable potency while ISIS 556846showed improvement in potency with comparable selectivity.

TABLE 105 Gap-interrupted modified oligonucleotides  targeting HTT SNPWing SEQ ISIS Sequence Gap chemistry ID NO. (5′ to 3′) chemistry 5′ 3′NO. 460209 T_(e)A_(k)A_(k)ATTGT Full  ekk kke 10 ^(m)CAT^(m)CA_(k)^(m)C_(k) ^(m)C_(e) deoxy 556845 T_(e)A_(k)A_(k)A^(x)TTGT Deoxy/ ekk kke10 ^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 2-Thio 556846T_(e)A_(k)A_(k)AT^(x)TGT Deoxy/ ekk kke 10 ^(m)CAT^(m)CA_(k) ^(m)C_(k)^(m)C_(e) 2-Thio 556847 T_(e)A_(k)A_(k)A^(x)T^(x)TGT Deoxy/ ekk kke 10^(m)CAT^(m)CA_(k) ^(m)C_(k) ^(m)C_(e) 2-Thio e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

TABLE 106 Comparison of inhibition of HTT mRNA levels and selectivity ofgap-interrupted modified oligonucleotides with ISIS 460209 targeting HTTSNP ISIS IC₅₀ (μM) Selectivity Gap Wing Chemistry NO Mut Wt (wt vs mut)chemistry 5′ 3′ 460209 0.30 0.99 3.3 Full deoxy ekk kke 556845 0.1310.01 >77 Deoxy/2-Thio ekk kke 556846 0.19 0.48 2.5 Deoxy/2-Thio ekk kke556847 0.45 9.9 >22 Deoxy/2-Thio ekk kke e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

Example 69 Evaluation of Modified Oligonucleotides Targeting HTT SNP—InVivo Study

Additional modified oligonucleotides were selected and tested for theireffects on mutant and wild type HTT protein levels in vivo targetingvarious SNP sites as illustrated below.

The gapmers and their motifs are described in Table 107. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt).

The gapmer, ISIS 460209 was included in the study as a benchmarkoligonucleotide against which the potency and selectivity of themodified oligonucleotides could be compared. A non-allele specificoligonucleotide, ISIS 387898, was used as a positive control.

Hu97/18 mice, the first murine model of HD that fully geneticallyrecapitulates human HD were used in the study. They were generated inHayden's lab by cross bred BACHD, YAC 18 and Hdh (−/−) mice.

Hu97/18 mice were treated with 300 μg of modified oligonucleotides by asingle unilateral intracerebroventricular (ICV) bolus injection. Thistreatment group consisted of 4 animals/oligonucleotide. The controlgroup received a 10 μl bolus injection of sterile PBS and consisted of 4animals.

Animals were sacrificed at 4 weeks post-injection. The second mostanterior 2 mm coronal slab for each brain hemisphere was collected usinga 2 mm rodent brain matrix. The remaining portion of the brain waspost-fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose andsectioned into 25 μm coronal sections for immunohistochemical analysis.

The HTT protein levels were analyzed by high molecular weight westernblot (modified from Invitrogen's NuPAGE Bis-Tris System Protocol). Thetissue was homogenized in ice cold SDP lysis buffer. 40 μg of totalprotein lysate was resolved on 10% low-BIS acrylamide gels (200:1acrylamide:BIS) with tris-glycine running buffer (25 mM Tris, 190 mMGlycince, 0.1% SDS) containing 10.7 mM β-mercaptoethanol added fresh.Gels were run at 90V for 40 min through the stack, then 190V for 2.5 h,or until the 75 kDa molecular weight marker band was at the bottom ofthe gel. Proteins were transferred to nitrocellulose at 24V for 2 h withNuPage transfer buffer (Invitrogen: 25 mM Bicine, 25 mM Bis-Tris, 1.025mM EDTA, 5% MeOH, pH 7.2). Membranes were blocked with 5% milk in PBS,and then blotted for HTT with MAB2166 (1:1000, millipore). Anti-calnexin(Sigma C4731) immunoblotting was used as loading control. Proteins weredetected with IR dye 800CW goat anti-mouse (Rockland 610-131-007) andAlexaFluor 680 goat anti-rabbit (Molecular Probes A21076)-labeledsecondary antibodies, and the LiCor Odyssey Infrared Imaging system.

The results in Table 108 are presented as the average percent of HTTprotein levels for each treatment group, normalized to PBS-treatedcontrol and is denoted as “% UTC”. The percent of mutant HTT proteinlevels is denoted as “mut”. The percent of wild-type HTT protein levelsis denoted as “wt”. Selectivity was also evaluated and measured bydividing the percent of wild-type HTT protein levels vs. the percent ofthe mutant HTT protein levels.

As illustrated in Table 108, treatment with the newly designedoligonucleotides, ISIS 476333 and 460085 showed improvement in potencyand selectivity in inhibiting mutant HTT protein levels as compared tothe parent gapmer, 460209. Comparable or a slight loss in potency and/orselectivity was observed for the remaining oligonucleotides.

TABLE 107 Modified oligonucleotides targeting HTT rs7685686,rs4690072 and rs363088 in Hu97/18 mice Wing SEQ ISIS Chemistry ID NOSequence (5′ to 3′) Motif 5′ 3′ NO. 387898C_(e)T_(e)C_(e)G_(e)A_(e)CTAAAGCAGGA_(e)T_(e)T_(e)T_(e)C_(e) 5-10-5 e5e5 79 460209 T_(e)A_(k)A_(k)ATTGTCATCA_(k)C_(k)C_(e) 3-9-3 ekk kke 10435879 AeA_(e)T_(e)A_(e)A_(e)ATTGTCATCA_(e)C_(e)C_(e)A_(e)G_(e) 5-9-5 e5e5 80 476333 A_(e)T_(k)A_(e)A_(k)ATTGTCATCA_(k)C_(e)CkA_(e) 4-9-4 ekekkeke 32 435874C_(e)A_(e)C_(e)A_(e)G_(e)TGCTACCCAA_(e)C_(e)C_(e)T_(e)T_(e) 5-9-5 e5 e581 435871 T_(e)C_(e)A_(e)C_(e)A_(e)GCTATCTTCT_(e)C_(e)A_(e)T_(e)C_(e)5-9-5 e5 e5 82 460085A_(e)T_(e)A_(e)A_(e)A_(e)TTGTCATC_(e)A_(e)C_(e)C_(e)A_(e) 5-7-5 e5 e5 32e = 2′-MOE (e.g. e5 = eeeee), k = cEt

TABLE 108 Effects of modified oligonucleotides on mutant and wild typeHTT protein levels in Hu97/18 mice Dosage % UTC Selectivity ISIS NO SNPsite (μg) mut wt (wt vs mut) PBS — 300 100 100 1 387898 — 300 23.7625.66 1 460209 rs7685686 300 18.16 48.99 2.7 435879 rs7685686 300 41.4873.11 1.8 476333 rs7685686 300 6.35 22.05 3.5 460085 rs7685686 300 2.940.1 13.8 435874 rs4690072 300 44.18 76.63 1.7 435871 rs363088 300 33.0789.30 2.7

Example 70 Evaluation of ISIS 435871 in Central Nervous System (CNS)Targeting HTT Rs363088—In Vivo Study

A modified oligonucleotide from Example 68, ISIS 435871 was selected andtested for its effects on mutant and wild type HTT protein levels in theCNS in vivo targeting rs363088.

Hu97/18 mouse was treated with 300 μg of ISIS 435871 by a singleunilateral intracerebroventricular (ICV) bolus injection. The animal wassacrificed at 4 weeks post-injection. Regional CNS structures were thenmicro-dissected including bilateral samples from the most anteriorportion of cortex (Cortex 1), an intermediate section of cortex (Cortex2), the most posterior section of cortex (Cortex 3), the striatum, thehippocampus, the cerebellum, and a 1 cm section of spinal cord directlybelow the brain stem. Tissue was homogenized and assessed for mutant andwild-type HTT levels by Western blotting using the procedures asdescribed in Example 69. The results are presented below. As nountreated or vehicle treated control is shown, HTT intensity of eachallele is expressed as a ratio of calnexin loading control intensity.The ratio of the mutant HTT to the wt HTT in the treated animal wasdetermined and is denoted as “wt/mut”. Having a ratio higher than 1 isindicative of allele-specific silencing.

As illustrated in Table 109, a single unilateral ICV bolus injection ofthe modified antisense oligonucleotide showed selective HTT silencingthroughout the CNS except in the cerebellum, where the antisenseoligonucleotide did not distribute evenly.

TABLE 109 Effects of ISIS 435871 on mutant and wild type HTT proteinlevels in CNS targeting rs363088 in Hu97/18 mice HTT intensity/calnexinintensity Tissue wt mut wt/mut Cortex 1 0.032 0.014 2.29 Cortex 2 0.0270.009 3 Cortex 3 0.023 0.007 3.29 Striatum 0.030 0.012 2.5 Hippocampus0.016 0.006 2.67 Cerebellum 0.023 0.019 1.21 Spinal Cord 0.014 0.007 2

Example 71 Evaluation of Modified Oligonucleotides Targeting HTTRs7685686—In Vivo Study

Several modified oligonucleotides from Examples 43, 51, 52, 53 and 66were selected and tested for their effects on mutant and wild type HTTprotein levels in vivo targeting HTT rs7685686.

The gapmer, ISIS 460209 was included in the study as a benchmarkoligonucleotide against which the potency and selectivity of themodified oligonucleotides could be compared.

Hu97/18 mice were treated with 300 μg of modified oligonucleotides by asingle unilateral intracerebroventricular (ICV) bolus injection. Thistreatment group consisted of 4 animals/oligonucleotide. The controlgroup received a 10 μl bolus injection of sterile PBS and consisted of 4animals.

Animals were sacrificed at 4 weeks post-injection. The second mostanterior 2 mm coronal slab for each brain hemisphere was collected usinga 2 mm rodent brain matrix. The HTT protein levels were analyzed in thesame manner as described in Example 69 and the results are presentedbelow.

The results in Table 110 are presented as the average percent of HTTprotein levels for each allele and treatment group, normalized toPBS-treated control and is denoted as “% UTC”. The percent of mutant HTTprotein levels is denoted as “mut”. The percent of wild-type HTT proteinlevels is denoted as “wt”.

As shown in Table 110, each of the newly designed oligonucleotidesshowed improvement in selective inhibition of mutant HTT protein levelsas compared to ISIS 460209. ISIS 550913 and 540095 showed improvement inpotency while the remaining modified oligonucleotides showed comparableor a slight decrease in potency as compared to the parent gapmer.

TABLE 110 Effects of modified oligonucleotides on mutant and wild typeHTT protein levels targeting rs7685686 in Hu97/18 mice Wing SEQ ISIS %UTC chemistry Gap ID NO mut wt Motif 5′ 3′ chemistry NO PBS 100 100 — —— — — 460209 18.16 48.99 3-9-3 ekk kke Full deoxy 10 550913 9.31 34.265-9-5 kkekk kkekk Full deoxy 27 540095 12.75 106.05 2-9-4 ek kkke Fulldeoxy 65 551429 19.07 108.31 5-7-3 eeekk kke Full deoxy 10 540094 24.6887.56 2-9-4 ek kkke Full deoxy 67 540096 24.89 98.26 2-9-4 ek kkke Fulldeoxy 68 540108 28.34 85.62 5-7-5 eeekk kkeee Full deoxy 23 e = 2′-MOE,k = cEt

Example 72 Evaluation of Modified Oligonucleotides Targeting HTTRs7685686—In Vivo Study

Several modified oligonucleotides selected from Examples 57, 58, 61 and62 were tested and evaluated for their effects on mutant and wild typeHTT protein levels in vivo targeting HTT rs7685686.

Hu97/18 mice were treated with 300 μg of modified oligonucleotides by asingle unilateral intracerebroventricular (ICV) bolus injection and thecontrol group received a 10 μl bolus injection of sterile PBS. Eachtreatment group consisted of 4 animals.

Animals were sacrificed at 4 weeks post-injection. The second mostanterior 2 mm coronal slab for each brain hemisphere was collected usinga 2 mm rodent brain matrix. The HTT protein levels were analyzed in thesame manner as described in Example 69. The in vivo study for ISIS575008 and 571069 marked with an asterisk (*) was performedindependently and the results are presented below.

The results in Table 111 are presented as the average percent of HTTprotein levels for each allele and treatment group, normalized toPBS-treated control and is denoted as “% UTC”. The percent of mutant HTTprotein levels is denoted as “mut”. The percent of wild-type HTT proteinlevels is denoted as “wt”.

As illustrated in Table 111, selective inhibition of mut HTT proteinlevels was achieved with the newly designed oligonucleotide treatment ascompared to PBS treated control.

TABLE 111 Effects of modified oligonucleotides on mutant and wild typeHTT protein levels targeting rs7685686 in Hu97/18 mice Wing SEQ ISIS %UTC chemistry Gap ID NO mut wt Motif 5′ 3′ chemistry NO PBS 100 100 — —— — — 575007 26.9 104.5 3-9-3 ekk kke Deoxy/cEt 10  575008* 21.7 105.95-7-3 ekkkk kke Deoxy/cEt 10 566267 32.8 109.3 3-9-3 ekk kke Deoxy/F- 10HNA 571036 30.3 103.3 6-7-4 ekekek keke Full deoxy 32 571037 32.8 111.96-7-4 eeeekk keke Full deoxy 32  571069* 29.4 109.8 6-7-4 eeeekk kkeeFull deoxy 32 e = 2′-MOE, k = cEt

Example 73 Evaluation of Modified Oligonucleotides Targeting HTTRs7685686—In Vivo Dose Response Study

ISIS 476333, 435871, 540108, 575007 and 551429 from previous exampleswere selected and evaluated at various doses for their effect on mutantand wild type HTT protein levels in vivo targeting HTT rs7685686.

Hu97/18 mice were treated with various doses of modifiedoligonucleotides as presented in Table 112 by a single unilateralintracerebroventricular (ICV) bolus injection. This treatment groupconsisted of 4 animals/oligonucleotide. The control group received a 10μl bolus injection of sterile PBS and consisted of 4 animals.

Animals were sacrificed at 4 weeks post-injection. The second mostanterior 2 mm coronal slab for each brain hemisphere was collected usinga 2 mm rodent brain matrix. The HTT protein levels were analyzed in thesame manner as described in Example 69. The dose response study wasperformed independently for each modified oligonucleotide and theresults are presented below.

The results in Table 112 are presented as the average percent of HTTprotein levels for each allele and treatment group, normalized toPBS-treated control and is denoted as “% UTC”. The percent of mutant HTTprotein levels is denoted as “mut”. The percent of wild-type HTT proteinlevels is denoted as “wt”.

As illustrated in Table 112, selective inhibition of mut HTT proteinlevels was achieved in a dose-dependent manner for the newly designedoligonucleotides.

TABLE 112 Dose-dependent effect of modified oligonucleotides on mutantand wild type HTT protein levels targeting rs7685686 in Hu97/18 miceDosage % UTC SEQ ISIS NO (μg) mut wt Motif ID NO. PBS 0 100 100 — 47633350 48.7 115 4-9-4 32 150 23.1 53.3 (ekek-d9-keke) 300 8.8 36.7 435871 75114 118 5-9-5 82 150 47.3 80.3 (e5-d9-e5) 300 33 89.3 500 36 97.5 54010875 30.5 71.7 5-7-5 32 150 22 81 (eeekk-d7-kkeee) 300 8.6 59.6 575007 15041.5 110.7 3-9-3 10 300 29 119.4 (ekk-d-k-d7-kke) (deoxy gap interruptedwith cEt) 551429 75 58 101.3 5-7-3 10 150 36.2 110.4 (eeekk-d7-kke) 30019.7 107.8 e = 2′-MOE (e.g. e5 = eeeee), k = cEt, d =2′-deoxyribonucleoside

Example 74 Modified Oligonucleotides Targeting Huntingtin (HTT) SingleNucleotide Polymorphism (SNP)

A series of modified oligonucleotides was designed based on a parentgapmer, ISIS 460209, wherein the central gap region contains nineβ-D-2′-deoxyribonucleosides. The modified oligonucleotides were designedby introducing a 5′-(R)-Me DNA modification within the central gapregion. The 5′-(R)-Me DNA containing oligonucleotides were tested fortheir ability to selectively inhibit mutant (mut) HTT mRNA expressionlevels targeting rs7685686 while leaving the expression of the wild-type(wt) intact. The potency and selectivity of the modifiedoligonucleotides were evaluated and compared to ISIS 460209.

The position on the oligonucleotides opposite to the SNP position, ascounted from the 5′-terminus is position 8.

The modified oligonucleotides were created with a 3-9-3 motif and aredescribed in Table 113. The internucleoside linkages throughout eachgapmer are phosphorothioate (P═S) linkages. Nucleosides followed by asubscript “d” are β-D-2′-deoxyribonucleosides. Nucleosides followed by asubscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside.Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH₃ bicyclicnucleoside (e.g. cEt). Nucleosides followed by a subscript “z” indicatesa 5′-(R)-Me DNA. “^(m)C” indicates a 5-methyl cytosine nucleoside.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith a single dose at 2 μM concentration of the modifiedoligonucleotide. After a treatment period of approximately 24 hours,cells were washed with DPBS buffer and lysed. RNA was extracted usingQiagen RNeasy purification and mRNA levels were measured by quantitativereal-time PCR using ABI assay C_(—)2229297_(—)10 which measures at dbSNPrs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCRbuffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RTMIX. To each well was added 15 uL of this mixture and 5 uL of purifiedRNA. The mutant and wild-type HTT mRNA levels were measuredsimultaneously by using two different fluorophores, FAM for mutantallele and VIC for wild-type allele. The HTT mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN.

The IC₅₀s and selectivities as expressed in “fold” were measured andcalculated using methods described previously in Example 41. Asillustrated in Table 114, treatment with the newly designedoligonucleotides showed comparable or a slight increase in potencyand/or selectivity as compared to ISIS 460209.

TABLE 113 Gap-interrupted oligonucleotides comprising 5′-(R)-Me DNAtargeting HTT SNP Wing SEQ ISIS chemistry ID NO. Sequence (5′ to 3′)Gap chemistry 5′ 3′ NO. 460209 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) Full deoxy ekkkke 10 556848 T_(e)A_(k)A_(k)A_(z)T_(d)T_(d)G_(d)T_(d)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e)Deoxy/5′-(R)-Me DNA ekk kke 10 556849T_(e)A_(k)A_(k)A_(d)T_(z)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) Deoxy/5′-(R)-Me DNA ekk kke 10 556850T_(e)A_(k)A_(k)A_(d)T_(d)T_(z)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) Deoxy/5′-(R)-Me DNA ekk kke 10 e =2′-MOE, k = cEt

TABLE 114 Comparison of inhibition of HTT mRNA levels and selectivity ofgap- interrupted oligonucleotides with ISIS 460209 targeting HTT SNPIC₅₀ Wing ISIS (μM) Selectivity Gap chemistry NO. Mut Wt (wt vs mut)chemistry 5′ 3′ 460209 0.30 0.99 3.3 Full deoxy ekk kke 556848 0.15 0.64.0 Deoxy/5′-(R)- ekk kke Me DNA 556849 0.16 0.46 2.9 Deoxy/5′-(R)- ekkkke Me DNA 556850 0.33 0.96 2.9 Deoxy/5′-(R)- ekk kke Me DNA e = 2′-MOE,k = cEt

Example 75 Modified Oligonucleotides Comprising 5′-(R)- or 5′-(S)-Me DNAModification Targeting HTT SNP

A series of modified oligonucleotides was designed based on a parentgapmer, ISIS 460209, wherein the central gap region contains nineβ-D-2′-deoxyribonucleosides. The modified oligonucleotides were designedby introducing 5′-(S)- or 5′-(R)-Me DNA modification slightly upstreamor downstream (i.e. “microwalk”) within the central gap region. Thegapmers were created with a 3-9-3 motif and were tested for theirability to selectively inhibit mutant (mut) HTT mRNA expression. Thepotency and selectivity of the modified oligonucleotides were evaluatedand compared to ISIS 460209.

The position on the oligonucleotides opposite to the SNP position, ascounted from the 5′-terminus is position 8.

The modified oligonucleotides and their motifs are described in Table115. The internucleoside linkages throughout each gapmer arephosphorothioate (P═S) linkages. Nucleosides followed by a subscript “d”are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosidesfollowed by a subscript “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt). Nucleosides followed by a subscript “v” indicates a5′-(S)-Me DNA. Nucleosides followed by a subscript “z” indicates a5′-(R)-Me DNA. “^(m)C” indicates a 5-methyl cytosine nucleoside.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used. Cultured GM04022 cells at adensity of 25,000 cells per well were transfected using electroporationwith 0.1, 0.4, 1.1, 3.3 and 10 μM concentrations of modifiedoligonucleotides. After a treatment period of approximately 24 hours,cells were washed with DPBS buffer and lysed. RNA was extracted usingQiagen RNeasy purification and mRNA levels were measured by quantitativereal-time PCR using ABI assay C_(—)2229297_(—)10 which measures at dbSNPrs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCRbuffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RTMIX. To each well was added 15 uL of this mixture and 5 uL of purifiedRNA. The mutant and wild-type HTT mRNA levels were measuredsimultaneously by using two different fluorophores, FAM for mutantallele and VIC for wild-type allele. The HTT mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN and the resultsare presented below.

The IC₅₀s and selectivities as expressed in “fold” were measured andcalculated using methods described previously in Example 41. The resultsin Table 116 demonstrated that each of the newly designedoligonucleotides comprising 5′-(S)- or 5′-(R)-Me DNA within the centralgap region achieved improvement in potency and selectivity as comparedto the parent gapmer, ISIS 460209.

TABLE 115 Gap-interrupted oligonucleotides comprising 5′-(S)- or5′-(R)-Me DNA targeting HTT SNP Wing SEQ ISIS Gap Chemistry ID NOSequence (5′ to 3′) Motif Chemistry 5′ 3′ NO 460209T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Full deoxy ekk kke 10 589429T_(e)A_(k)A_(k)A_(d)T_(v)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(S)- ekk kke 10 Me DNA589430 T_(e)A_(k)A_(k)A_(d)T_(d)T_(v)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(S)- ekk kke 10 Me DNA589431 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(v) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(S)- ekk kke 10 Me DNA589432 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(v)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(S)- ekk kke 10 Me DNA594588 T_(e)A_(k)A_(k)A_(d)T_(v)T_(v)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(S)- ekk kke 10 Me DNA556848 T_(e)A_(k)A_(k)A_(z)T_(d)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10 Me DNA556849 T_(e)A_(k)A_(k)A_(d)T_(z)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10 Me DNA556850 T_(e)A_(k)A_(k)A_(d)T_(d)T_(z)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10 Me DNA539558 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(z) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10 Me DNA594160 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(z)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10 Me DNA594161 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(d)A_(z)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10 Me DNA589433 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(z)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10 Me DNA594162 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(z)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10 Me DNA594589 T_(e)A_(k)A_(k)A_(d)T_(z)T_(z)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10 Me DNAe = 2′-MOE; k = cEt

TABLE 116 Comparison of inhibition of HTT mRNA levels and selectivity ofgap- interrupted oligonucleotides with ISIS 460209 targeting HTT SNPISIS IC₅₀ (μM) Selectivity Wing Chemistry NO. Mut Wt (wt vs. mut) MotifGap Chemistry 5′ 3′ 460209 1.2 1.4 1.2 3-9-3 Full deoxy ekk kke 5894290.22 3.3 15 3-9-3 Deoxy/5′-(S)-Me DNA ekk kke 589430 0.22 >10 >45.53-9-3 Deoxy/5′-(S)-Me DNA ekk kke 589431 0.16 1.9 11.9 3-9-3Deoxy/5′-(S)-Me DNA ekk kke 589432 0.23 >10 >43.5 3-9-3 Deoxy/5′-(S)-MeDNA ekk kke 594588 0.81 >10 >12.3 3-9-3 Deoxy/5′-(S)-Me DNA ekk kke556848 0.16 1.8 11.3 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke 556849 0.14 1.17.9 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke 556850 0.22 1.7 7.7 3-9-3Deoxy/5′-(R)-Me DNA ekk kke 539558 0.38 3.8 10 3-9-3 Deoxy/5′-(R)-Me DNAekk kke 594160 0.28 3.3 11.8 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke 5941610.28 >10 >35.7 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke 589433 0.27 4.4 16.33-9-3 Deoxy/5′-(R)-Me DNA ekk kke 594162 0.27 3.5 13.0 3-9-3Deoxy/5′-(R)-Me DNA ekk kke 594589 0.48 4.4 9.2 3-9-3 Deoxy/5′-(R)-MeDNA ekk kke e = 2′-MOE; k = cEt

Example 76 Inhibition of HTT mRNA Levels Targeting SNP by ModifiedOligonucleotides

Additional modified oligonucleotides were designed in a similar manneras the antisense oligonucleotides described in Example 75. Variouschemical modifications were introduced slightly upstream or downstream(i.e. “microwalk”) within the central gap region. The gapmers werecreated with a 3-9-3 motif and were tested for their ability toselectively inhibit mutant (mut) HTT mRNA expression. The position onthe oligonucleotides opposite to the SNP position, as counted from the5′-terminus is position 8. The potency and selectivity of the modifiedoligonucleotides were evaluated and compared to ISIS 460209.

The modified oligonucleotides and their motifs are described in Table117. The internucleoside linkages throughout each gapmer arephosphorothioate (P═S) linkages. Nucleosides followed by a subscript “d”are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”indicates a 2′-β-methoxyethyl (MOE) modified nucleoside. Nucleosidesfollowed by a subscript “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt). Nucleosides followed by a subscript “b” indicates a5′-(R)-allyl DNA. Nucleosides followed by a subscript “c” indicates a5′-(S)-allyl DNA. Nucleosides followed by a subscript “g” indicates a5′-(R)-hydroxyethyl DNA. Nucleosides followed by a subscript “i”indicates a 5′-(S)-hydroxyethyl DNA. “^(m)C” indicates a 5-methylcytosine nucleoside.

The modified oligonucleotides were tested in vitro using heterozygousfibroblast GM04022 cell line. The transfection method and analysis ofHTT mRNA levels adjusted according to total RNA content, as measured byRIBOGREEN were performed in the same manner as described in Example 76.The IC₅₀s and selectivities as expressed in “fold” were measured andcalculated using methods described previously and the results are shownbelow. As presented in Table 118, several modified oligonucleotidesachieved greater than 4.5 fold selectivity in inhibiting mutant HTT mRNAlevels and, therefore, are more selective than ISIS 460209.

TABLE 117 Gap-interrupted oligonucleotides comprising 5′-substituted DNAtargeting HTT SNP Wing SEQ ISIS Gap Chemistry Chemisty ID NOSequence (5′ to 3′) Motif (mod position) 5′ 3′ NO 460209T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Full deoxy ekk kke 10 589414T_(e)A_(k)A_(k)A_(d)T_(b)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10allyl DNA (pos 5) 589415 T_(e)A_(k)A_(k)A_(d)T_(d)T_(b)G_(d)T_(d)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/5′-(R)- ekk kke 10 allyl DNA (pos 6) 589416T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(b) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10allyl DNA (pos 8) 589417 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d)^(m)C_(d)A_(d)T_(b) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/5′-(R)- ekk kke 10 allyl DNA (pos 11) 589418T_(e)A_(k)A_(k)A_(d)T_(c)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(S)- ekk kke 10allyl DNA (pos 5) 589419 T_(e)A_(k)A_(k)A_(d)T_(d)T_(c)G_(d)T_(d)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/5′-(S)- ekk kke 10 allyl DNA (pos 6) 589420T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(c) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(S)- ekk kke 10allyl DNA (pos 8) 589421 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d)^(m)C_(d)A_(d)T_(c) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/5′-(S)- ekk kke 10 allyl DNA (pos 11) 589422T_(e)A_(k)A_(k)A_(d)T_(g)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10hydroxyethyl DNA (pos 5) 589423 T_(e)A_(k)A_(k)A_(d)T_(d)T_(g)G_(d)T_(d)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/5′-(R)- ekk kke 10 hydroxyethyl DNA (pos 6) 589424T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(g) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(R)- ekk kke 10hydroxyethyl DNA (pos 8) 589437 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d)^(m)C_(d)A_(d)T_(g) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/5′-(R)- ekk kke 10 hydroxyethyl DNA (pos 11) 589426T_(e)A_(k)A_(k)A_(d)T_(i)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/5′-(S)- ekk kke 10hydroxyethyl DNA (pos 5) 589427 T_(e)A_(k)A_(k)A_(d)T_(d)T_(i)G_(d)T_(d)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/5′-(S)- ekk kke 10 hydroxyethyl DNA (pos 6) 589428T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(i) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C^(e) 3-9-3 Deoxy/5′-(S)- ekk kke 10hydroxyethyl DNA (pos 8) 589425 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d)^(m)C_(d)A_(d)T_(i) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/5′-(S)- ekk kke 10 hydroxyethyl DNA (pos 11) e = 2′-MOE; k = cEt

TABLE 118 Comparison of inhibition of HTT mRNA levels and selectivity ofgap- interrupted oligonucleotides with ISIS 460209 targeting HTT SNPISIS IC₅₀ (μM) Selectivity Gap Chemistry Wing Chemistry NO Mut Wt (wtvs. mut) (mod position) Motif 5′ 3′ 460209 0.47 2.1 4.5 Full deoxy 3-9-3ekk kke 589414 1.0 7.6 7.6 Deoxy/5′-(R)-Allyl DNA 3-9-3 ekk kke (pos 5)589415 1.4 >10 >7.1 Deoxy/5′-(R)-Allyl DNA 3-9-3 ekk kke (pos 6) 5894162.7 >10 >3.7 Deoxy/5′-(R)-Allyl DNA 3-9-3 ekk kke (pos 8) 5894175.4 >10 >1.9 Deoxy/5′-(R)-Allyl DNA 3-9-3 ekk kke (pos 11) 5894181.2 >10 >8.3 Deoxy/5′-(S)-Allyl DNA 3-9-3 ekk kke (pos 5) 5894191.1 >10 >9.1 Deoxy/5′-(S)-Allyl DNA 3-9-3 ekk kke (pos 6) 5894203.2 >10 >3.1 Deoxy/5′-(S)-Allyl DNA 3-9-3 ekk kke (pos 8) 5894212.0 >10 >5.0 Deoxy/5′-(S)-Allyl DNA 3-9-3 ekk kke (pos 11) 589422 0.733.2 4.4 Deoxy/5′-(R)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 5) 589423 0.929.2 10 Deoxy/5′-(R)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 6) 589424 0.214.4 21 Deoxy/5′-(R)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 8) 5894370.73 >10.2 >14 Deoxy/5′-(R)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 11)589426 0.91 5.1 5.6 Deoxy/5′-(5> 3-9-3 ekk kke Hydroxyethyl DNA (pos 5)589427 0.91 >10 >11 Deoxy/5′-(S)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 6)589428 1.1 >11 >10 Deoxy/5′-(S)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 8)589425 1.5 >10.5 >7 Deoxy/5′-(S)- 3-9-3 ekk kke Hydroxyethyl DNA (pos11) e = 2′-MOE; k = cEt

Example 77 Modified Oligonucleotides Comprising 5′-(R)-Me DNA(s)Targeting Human C-Reactive Protein (hCRP)

A series of modified oligonucleotides were designed based on ISIS353512, wherein the central gap region contains fourteenβ-D-2′-deoxyribonucleoside. These modified oligonucleotides weredesigned by replacement of two or three β-D-2′-deoxyribonucleoside inthe 14 nucleoside gap region with 5′-(R)-Me DNA(s). The thermalstability (T_(m)) and potency of these modified oligonucleotidestargeting hCRP was evaluated. The 3-14-3 MOE gapmer, ISIS 353512 and5-10-5 MOE gapmer, ISIS 330012 were included in the study forcomparison.

The modified oligonucleotides and their motifs are described in Table119. Each internucleoside linkage is a phosphorothioate (P═S) except fornucleosides followed by a subscript “o”which are phosphodiesterinternucleoside linkages (P═O). Nucleosides followed by a subscript “d”indicates a β-D-2′-deoxyribonucleoside. Nucleosides followed by asubscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside.Nucleosides followed by a subscript “z” indicates a 5′-(R)-Me DNA.“^(m)C” indicates a 5-methyl cytosine modified nucleoside. Underlinednucleosides indicate a region comprising 5′-(R)-Me DNA modification.

Thermal Stability Assay

The modified oligonucleotides were evaluated in thermal stability(T_(m)) assay. The T_(m)'s were measured using the method describedherein. A Cary 100 Bio spectrophotometer with the Cary Win UV Thermalprogram was used to measure absorbance vs. temperature. For the T_(m)experiments, oligonucleotides were prepared at a concentration of 8 μMin a buffer of 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, pH 7.Concentration of oligonucleotides were determined at 85° C. Theoligonucleotide concentration was 4 μM with mixing of equal volumes oftest oligonucleotide and complimentary RNA strand. Oligonucleotides werehybridized with the complimentary RNA strand by heating duplex to 90° C.for 5 min and allowed to cool at room temperature. Using thespectrophotometer, T_(m) measurements were taken by heating duplexsolution at a rate of 0.5 C/min in cuvette starting @ 15° C. and heatingto 85° C. T_(m) values were determined using Vant Hoff calculations(A₂₆₀ vs temperature curve) using non self-complementary sequences wherethe minimum absorbance which relates to the duplex and the maximumabsorbance which relates to the non-duplex single strand are manuallyintegrated into the program. The results are presented below.

Cell Culture and Transfection

The modified oligonucleotides were tested in vitro. Hep3B cells wereplated at a density of 40,000 cells per well and transfected usingelectroporation with 0.009 μM, 0.027 μM, 0.082 μM, 0.25 μM, 0.74 μM, 2.2μM, 6.7 μM and 20 μM concentrations of antisense oligonucleotides. Aftera treatment period of approximately 16 hours, RNA was isolated from thecells and hCRP mRNA levels were measured by quantitative real-time PCR.Human CRP primer probe set RTS1887 was used to measure mRNA levels. hCRPmRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®.

Analysis of IC₅₀'s

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis presented below and was calculated by plotting the concentrations ofoligonucleotides used versus the percent inhibition of hCRP mRNAexpression achieved at each concentration, and noting the concentrationof oligonucleotide at which 50% inhibition of hCRP mRNA expression wasachieved compared to the control.

As illustrated in Table 120, treatment with the newly designedoligonucleotides showed no improvement in potency as compared to thecontrols, ISIS 353512 and 330012.

TABLE 119 Gap-interrupted oligonucleotides comprising 5′-(R)-Me DNA targeting hCRP Wing SEQ ISIS Gap Chemistry Linkage ID NO Sequence (5′to 3′) Motif Chemistry 5′ 3′ backbone NO 353512 T_(e) ^(m)C_(e)^(m)C_(e) ^(m)C_(d)A_(d)T_(d)T_(d)T_(d) ^(m)C_(d)A_(d) 3-14-3 Full deoxyeee eee Full PS 83 G_(d)G_(d)A_(d)G_(d)A_(d) ^(m)C_(d)^(m)C_(d)T_(e)G_(e)G_(e) 546127 T_(e) ^(m)C_(e) ^(m)C_(e)^(m)C_(d)A_(d)T_(d)T_(d)T_(d) ^(m)C_(do) A_(zo) 3-14-3 Deoxy/5′-(R)- eeeeee Mixed 83 G_(z) G_(d)A_(d)G_(d)A_(d) ^(m)C_(d)^(m)C_(d)T_(e)G_(e)G_(e) Me DNA PS/PO 544810 T_(e) ^(m)C_(e) ^(m)C_(e)^(m)C_(d)A_(d)T_(d)T_(d)T_(d) ^(m)C_(d)A_(d) 3-14-3 Deoxy/5′-(R)- eeeeee Mixed 83 G_(d)G_(d)A_(d)G_(d)A_(do) ^(m)C_(zo) ^(m)C_(z)T_(e)G_(e)G_(e) Me DNA PS/PO 544806 T_(e) ^(m)C_(e) ^(m)C_(eo)^(m)C_(zo)A_(zo)T_(z) T_(d)T_(d) ^(m)C_(d)A_(d) 3-14-3 Deoxy/5′-(R)- eeeeee Mixed 83 G_(d)G_(d)A_(d)G_(d)A_(d) ^(m)C_(d)^(m)C_(d)T_(e)G_(e)G_(e) Me DNA PS/PO 544807 T_(e) ^(m)C_(e) ^(m)C_(e)^(m)C_(d)A_(d)T_(do) T_(zo)T_(zo) ^(m)C_(z) A_(d) 3-14-3 Deoxy/5′-(R)-eee eee Mixed 83 G_(d)G_(d)A_(d)G_(d)A_(d) ^(m)C_(d)^(m)C_(d)T_(e)G_(e)G_(e) Me DNA PS/PO 544809 T_(e) ^(m)C_(e) ^(m)C_(e)^(m)C_(d)A_(d)T_(d)T_(d)T_(d) ^(m)C_(d)A_(d) 3-14-3 Deoxy/5′-(R)- eeeeee Mixed 83 G_(d)G_(do) A_(zo)G_(zo)A_(z) ^(m)C_(d)^(m)C_(d)T_(e)G_(e)G_(e) Me DNA PS/PO 330012 T_(e) ^(m)C_(e) ^(m)C_(e)^(m)C_(e)A_(e)T_(d)T_(d)T_(d) ^(m)C_(d)A_(d) 5-10-5 Full deoxy e5 e5Full PS 83 G_(d)G_(d)A_(d)G_(d)A_(d) ^(m)C_(e) ^(m)C_(e)T_(e)G_(e)G_(e)e = 2′-MOE (e.g. e5 = eeeee)

TABLE 120 Effect of gap-interrupted oligonucleotide treatment on Tm andhCRP inhibition Wing ISIS Tm IC₅₀ Gap Chemistry Linkage NO (° C.) (μM)Motif Chemistry 5′ 3′ backbone 353512 66.7 1.1 3-14-3 Full deoxy eee eeeFull PS 546127 65.9 2.5 3-14-3 Deoxy/5′-(R)- eee eee Mixed Me DNA PS/PO544810 64.3 2.4 3-14-3 Deoxy/5′-(R)- eee eee Mixed Me DNA PS/PO 54480662.8 2.8 3-14-3 Deoxy/5′-(R)- eee eee Mixed Me DNA PS/PO 544807 65.1 2.73-14-3 Deoxy/5′-(R)- eee eee Mixed Me DNA PS/PO 544809 64.2 5.0 3-14-3Deoxy/5′-(R)- eee eee Mixed Me DNA PS/PO 330012 71.7 0.6 5-10-5 Fulldeoxy e5 e5 Full PS e = 2′-MOE (e.g. e5 = eeeee), PS/PO =phosphorothioate/phosphodiester internucleoside linkage

Example 78 Human Peripheral Blood Mononuclear Cells (hPBMC) AssayProtocol—In Vitro

The hPBMC assay was performed using BD Vacutainer CPT tube method. Asample of whole blood from volunteered donors with informed consent atUS HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtainedand collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753).The approximate starting total whole blood volume in the CPT tubes foreach donor was recorded using the PBMC assay data sheet.

The blood sample was remixed immediately prior to centrifugation bygently inverting tubes 8-10 times. CPT tubes were centrifuged at rt(18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800RCF with brake off (2700 RPM Beckman Allegra 6R). The cells wereretrieved from the buffy coat interface (between Ficoll and polymer gellayers); transferred to a sterile 50 ml conical tube and pooled up to 5CPT tubes/50 ml conical tube/donor. The cells were then washed twicewith PBS (Ca⁺⁺, Mg⁺⁺ free; GIBCO). The tubes were topped up to 50 ml andmixed by inverting several times. The sample was then centrifuged at330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) andaspirated as much supernatant as possible without disturbing pellet. Thecell pellet was dislodged by gently swirling tube and resuspended cellsin RPMI+10% FBS+pen/strep (˜1 ml/10 ml starting whole blood volume). A60 μl sample was pipette into a sample vial (Beckman Coulter) with 600μl VersaLyse reagent (Beckman Coulter Cat# A09777) and was gentlyvortexed for 10-15 sec. The sample was allowed to incubate for 10 min.at rt and being mixed again before counting. The cell suspension wascounted on Vicell XR cell viability analyzer (Beckman Coulter) usingPBMC cell type (dilution factor of 1:11 was stored with otherparameters). The live cell/ml and viability were recorded. The cellsuspension was diluted to 1×10⁷ live PBMC/ml in RPMI+10% FBS+pen/strep.

The cells were plated at 5×10⁵ in 50 μl/well of 96-well tissue cultureplate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controlsdiluted in RPMI+10% FBS+pen/strep. was added according to experimenttemplate (100 μl/well total). Plates were placed on the shaker andallowed to mix for approx. 1 min. After being incubated for 24 hrs at37° C.; 5% CO₂, the plates were centrifuged at 400×g for 10 minutesbefore removing the supernatant for MSD cytokine assay (i.e. human IL-6,IL-10, IL-8 and MCP-1).

Example 79 Evaluation of the Proinflammatory Effects in hPBMC Assay for5′-(R)-Me DNA Containing Modified Oligonucleotides—In Vitro Study

The modified oligonucleotides targeting hCRP from Example 77 were testedand evaluated for the proinflammatory response in hPBMC assay usingmethods described previously in Example 78. The hPBMCs were isolatedfrom fresh, volunteered donors and were treated with modifiedoligonucleotides at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 μMconcentrations using the hPBMC assay protocol described herein. After a24 hr treatment, the cytokine levels were measured.

IL-6 was used as the primary readout. The resulting IL-6 level wascompared to the positive control, ISIS 353512 and negative control, ISIS104838. The results are presented in Table 121. As illustrated,reduction in proinflammatory response was achieved with the newlydesigned oligonucleotides at doses evaluated as compared to the positivecontrol, ISIS 353512.

ISIS 104838 designated herein as SEQ ID NO: 84, is a 5-10-5 MOE gapmerwith the following sequence, G_(e)^(m)C_(e)T_(e)G_(e)A_(e)T_(d)T_(d)A_(d)G_(d)A_(d)G_(d)A_(d)G_(d)A_(d)G_(d)G_(e)T_(e)^(m)C_(e) ^(m)C_(e) ^(m)C_(e). Each internucleoside linkage is aphosphorothioate (P═S). Each nucleoside followed by a subscript “d” is aβ-D-2′-deoxyribonucleoside. Each “^(m)C” is a 5-methyl cytosine modifiednucleoside and each nucleoside followed by a subscript “e” is a2′-O-methoxyethyl(MOE) modified nucleoside.

TABLE 121 Effect of gap-interrupted oligonucleotide treatment onproinflammatory response in hPBMC Wing ISIS Conc. IL-6 Gap ChemistryLinkage NO (uM) (pg/mL) Motif Chemistry 5′ 3′ backbone 353512 0 26.93-14-3 Full deoxy eee eee Full PS (pos 0.0128 10.6 control) 0.064 73.30.32 219.8 1.6 200.1 8 287.8 40 376.9 200 181.5 546127 0 11.5 3-14-3Deoxy/5′-(R)- eee eee Mixed 0.0128 15.1 Me DNA PS/PO 0.064 19.0 0.3237.3 1.6 67.5 8 86.3 40 111.2 200 83.1 544810 0 11.5 3-14-3Deoxy/5′-(R)- eee eee Mixed 0.0128 13.9 Me DNA PS/PO 0.064 15.1 0.3224.9 1.6 34.0 8 66.2 40 96.8 200 76.5 06/544806 0 11.3 3-14-3Deoxy/5′-(R)- eee eee Mixed 0.0128 10.8 Me DNA PS/PO 0.064 25.8 0.3215.6 1.6 25.4 8 52.3 40 69.3 200 341.7 06/544807 0 13.3 3-14-3Deoxy/5′-(R)- eee eee Mixed 0.0128 13.7 Me DNA PS/PO 0.064 18.4 0.3253.3 1.6 18.4 8 164.9 40 202.7 200 606.5 06/544809 0 10.8 3-14-3Deoxy/5′-(R)- eee eee Mixed 0.0128 13.3 Me DNA PS/PO 0.064 14.3 0.3234.8 1.6 62.3 8 100.9 40 213.1 200 225.0 06/330012 0 10.9 5-10-5 Fulldeoxy e5 e5 Full PS 0.0128 12.9 0.064 10.8 0.32 25.3 1.6 44.2 8 87.5 4080.2 200 82.3 07/104838 0 9.3 5-10-5 Full deoxy e5 e5 Full PS (neg0.0128 10.4 control) 0.064 17.6 0.32 30.1 1.6 53.9 8 124.8 40 94.5 20089.3 e = 2′-MOE (e.g. e5 = eeeee)

Example 80 Evaluation of the Proinflammatory Effects in hPBMC Assay fora Modified Oligonucleotide Comprising Methyl ThiophosphonateInternucleoside Linkages—In Vitro Study

A modified oligonucleotide was designed based on the 3/14/3 MOE gapmer,ISIS 353512. This modified oligonucleotide was created by havingalternating methyl thiophosphonate (—P(CH₃)(═S)—) internucleosidelinkages throughout the gap region. The proinflammatory effect of themodified oligonucleotide targeting hCRP was evaluated in hPBMC assayusing the protocol described in Example 78.

The modified oligonucleotide and its motif are described in Table 122.Each internucleoside linkage is a phosphorothioate (P═S) except fornucleosides followed by a subscript “w”. Each nucleoside followed by asubscript “w” indicates a methyl thiophosphonate internucleoside linkage(—P(CH₃)(═S)—). Nucleosides followed by a subscript “d” is aβ-D-2′-deoxyribonucleoside. Nucleosides followed by a subscript “e”indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. “^(m)C”indicates a 5-methyl cytosine modified nucleoside.

The hPBMCs were isolated from fresh, volunteered donors and were treatedwith modified oligonucleotides at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and200 μM concentrations. After a 24 hr treatment, the cytokine levels weremeasured.

IL-6 was used as the primary readout. The resulting IL-6 level wascompared to the positive control oligonucleotide, ISIS 353512 andnegative control, ISIS 104838. The results from two donors denoted as“Donor 1” and “Donor 2” are presented in Table 123. As illustrated,reduction in proinflammatory response was achieved with the newlydesigned oligonucleotide at doses evaluated as compared to the positivecontrol, ISIS 353512.

TABLE 122 Modified oligonucleotide comprising alternating methyl thiophosphonate internucleoside linkages throughout  the gap region WingSEQ ISIS Gap Chemistry ID NO Sequence (5′ to 3′) Motif Chemistry 5′ 3′NO 353512 T_(e) ^(m)C_(e) ^(m)C_(e) ^(m)C_(d)A_(d)T_(d)T_(d)T_(d)^(m)C_(d)A_(d)G_(d) 3-14-3 Full deoxy eee eee 83 G_(d)A_(d)G_(d)A_(d)^(m)C_(d) ^(m)C_(d)T_(e)G_(e)G_(e) 560221 T_(e) ^(m)C_(e)^(m)C_(e)C_(dw)A_(d)T_(dw)T_(d)T_(dw) ^(m)C_(d)A_(dw)G_(d)G_(dw) 3-14-3Deoxy/methyl eee eee 83 A_(d)G_(dw)A_(d)C_(dw) ^(m)C_(d)T_(e)G_(e)G_(e)thiophosphonate 104838 G_(e)^(m)C_(e)T_(e)G_(e)A_(e)T_(d)T_(d)A_(d)G_(d)A_(d)G_(d)A_(d) 5-10-5Full deoxy e5 e5 84 G_(d)A_(d)G_(d)G_(e)T_(e) ^(m)C_(e) ^(m)C_(e)^(m)C_(e) e = 2′-MOE (e.g. e5 = eeeee)

TABLE 123 Effect of modified oligonucleotide treatment onproinflammatory response in hPBMC assay Wing ISIS Conc. IL-6 (Donor 1)IL-6 (Donor 2) Gap Chemistry NO (μM) (pg/mL) (pg/mL) Motif Chemistry 5′3′ 353512 0 6.3 7.8 3-14-3 Full deoxy eee eee 0.0128 8.3 10.2 0.064 77.2118.2 0.32 151.9 394.3 1.6 152.4 395.3 8 147.6 337.2 40 122.5 228.4 200119.7 193.5 560221 0 5.6 7.6 3-14-3 Deoxy/methyl eee eee 0.0128 6.4 6.9thiophosphonate 0.064 6.7 7.6 0.32 7.6 8.9 1.6 9.1 11.8 8 17.5 24.3 4065.8 50.2 200 60.0 100.4 104838 0 5.8 7.3 5-10-5 Full deoxy e5 e5 0.01287.7 7.9 0.064 7.5 11.6 0.32 15.1 22.0 1.6 73.1 112.8 8 29.6 51.5 40 41.669.5 200 55.4 4018 e = 2′-MOE (e.g. e5 = eeeee)

Example 81 Modified Oligonucleotides Comprising Methyl PhosphonateInternucleoside Linkage Targeting HTT SNP—In Vitro Study

ISIS 558255 and 558256 from Example 49 were selected and evaluated fortheir effect on mutant and wild type HTT mRNA expression levelstargeting rs7685686. ISIS 46020 was included in the study forcomparison. The position on the oligonucleotides opposite to the SNPposition, as counted from the 5′-terminus is position 8.

Heterozygous fibroblast GM04022 cell line was used for the in vitroassay (from Coriell Institute). Cultured GM04022 cells at a density of25,000 cells per well were transfected using electroporation with 0.12,0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides.After a treatment period of approximately 24 hours, cells were washedwith DPBS buffer and lysed. RNA was extracted using Qiagen RNeasypurification and mRNA levels were measured by quantitative real-time PCRusing ABI assay C_(—)2229297_(—)10 which measures at dbSNP rs362303.RT-PCR method in short; A mixture was made using 2020 μL 2×PCR buffer,101 μL primers (300 μM from ABI), 1000 μL water and 40.4 μL RT MIX. Toeach well was added 15 μL of this mixture and 5 μL of purified RNA. Themutant and wild-type HTT mRNA levels were measured simultaneously byusing two different fluorophores, FAM for mutant allele and VIC forwild-type allele. The HTT mRNA levels were adjusted according to totalRNA content, as measured by RIBOGREEN.

The IC₅₀s and selectivities as expressed in “fold” were measured andcalculated using methods described previously in Example 41. Asillustrated in Table 124, improvement in selectivity and potency wasachieved with modified oligonucleotides comprising methyl phosphonateinternucleoside linkage as compared to ISIS 460209.

TABLE 124 Comparison of selectivity in inhition of HTT mRNA levels ofantisense oligonucleotides with ISIS 460209 targeted to rs7685686 inGM4022 cells ISIS IC₅₀ (μM) Selectivity Wing Chemistry SEQ NO Mut Wt (wtvs mut) Motif Gap Chemistry 5′ 3′ ID NO 460209 0.30 0.99 3.3 3-9-3 Fulldeoxy ekk kke 10 558255 0.19 1.3 6.8 3-9-3 Deoxy/Methyl ekk kke 10phosphonate 558256 0.20 1.3 6.5 3-9-3 Deoxy/Methyl ekk kke 10phosphonate e = 2′-MOE (e.g. e5 = eeeee), k = cEt

Example 82 Modified Oligonucleotides Comprising Methyl Phosphonate orPhosphonoacetate Internucleoside Linkage(s) Targeting HTT SNP

A series of modified oligonucleotides were designed based on ISIS 460209wherein the gap region contains nine β-D-2′-deoxyribonucleosides. Themodified oligonucleotides were synthesized to include one or more methylphosphonate or phosphonoacetate internucleoside linkage modificationswithin the gap region. The oligonucleotides with modified phosphoruscontaining backbone were tested for their ability to selectively inhibitmutant (mut) HTT mRNA expression levels targeting rs7685686 whileleaving the expression of the wild-type (wt) intact. The potency andselectivity of the modified oligonucleotides were evaluated and comparedto ISIS 460209.

The position on the oligonucleotides opposite to the SNP position, ascounted from the 5′-terminus is position 8.

The modified oligonucleotides and their motifs are described in Table125. Each internucleoside linkage is a phosphorothioate (P═S) except forthe internucleoside linkage having a subscript “x” or “y”. Eachnucleoside followed by a subscript “x” indicates a methyl phosphonateinternucleoside linkage (—P(CH₃)(═O)—). Each nucleoside followed by asubscript “y” indicates a phosphonoacetate internucleoside linkage(—P(CH₂CO₂ ⁻)(═O)—). Nucleosides followed by a subscript “d” is aβ-D-2′-deoxyribonucleoside. Nucleosides followed by a subscript “e”indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosidesfollowed by a subscript “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt). “^(m)C” indicates a 5-methyl cytosine modified nucleoside.

The modified oligonucleotides were tested in vitro. Heterozygousfibroblast GM04022 cell line was used (from Coriell Institute). CulturedGM04022 cells at a density of 25,000 cells per well were transfectedusing electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrationsof modified oligonucleotides. After a treatment period of approximately24 hours, cells were washed with DPBS buffer and lysed. RNA wasextracted using Qiagen RNeasy purification and mRNA levels were measuredby quantitative real-time PCR using ABI assay C_(—)2229297_(—)10 whichmeasures at dbSNP rs362303. RT-PCR method in short; A mixture was madeusing 2020 μL 2×PCR buffer, 101 μL primers (300 μM from ABI), 1000 uLwater and 40.4 μL RT MIX. To each well was added 15 μL of this mixtureand 5 μL of purified RNA. The mutant and wild-type HTT mRNA levels weremeasured simultaneously by using two different fluorophores, FAM formutant allele and VIC for wild-type allele. The HTT mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN.

The IC₅₀s and selectivities as expressed in “fold” were measured andcalculated using methods described previously in Example 41. Asillustrated in Table 126, most of the newly design oligonucleotidesachieved improvement in selectivity while maintaining potency ascompared to ISIS 460209.

TABLE 125 Modified oligonucleotides comprising methyl phosphonate orphosphonoacetate internucleoside linkage(s) targeting HTT SNP Wing SEQISIS Chemistry ID NO Sequence (5′ to 3′) Motif Gap Chemistry 5′ 3′ NO460209 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Full deoxy ekk kke 10 566276T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(dx)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/Methyl  ekk kke 10phosphonate 566277 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(dx)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/Methyl  ekk kke 10 phosphonate 566278T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(dx)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/Methyl  ekk kke 10phosphonate 566279 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d)^(m)C_(d)A_(dx)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/Methyl  ekk kke 10 phosphonate 566280T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(dx)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/Methyl  ekk kke 10phosphonate 566283 T_(e)A_(k)A_(k)A_(d)T_(dx)T_(dx)G_(d)T_(d)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3Deoxy/Methyl  ekk kke 10 phosphonate 573815T_(e)A_(k)A_(k)A_(d)T_(dy)T_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/ ekk kke 10Phosphonoacetate 573816 T_(e)A_(k)A_(k)A_(d)T_(d)T_(dy)G_(d)T_(d)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/ ekkkke 10 Phosphonoacetate 573817 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(dy)^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/ ekkkke 10 Phosphonoacetate 573818 T_(e)A_(k)A_(k)A_(d)T_(d)T_(d)G_(d)T_(d)^(m)C_(d)A_(d)T_(dy) ^(m)C_(d)A_(k) ^(m)C_(k) ^(m)C_(e) 3-9-3 Deoxy/ ekkkke 10 Phosphonoacetate e = 2′-MOE, k = cEt

TABLE 126 Comparison of selectivity in inhition of HTT mRNA levels ofantisense oligonucleotides with ISIS 460209 targeted to rs7685686 inGM4022 cells ISIS Mut IC₅₀ Selectivity Wing Chemistry SEQ NO (μM)) (wtvs mut) Motif Gap Chemistry 5′ 3′ ID NO 460209 0.15 9.4 3-9-3 Full deoxyekk kke 10 566276 0.76 12.8 3-9-3 Deoxy/Methyl phosphonate ekk kke 10566277 0.20 17 3-9-3 Deoxy/Methyl phosphonate ekk kke 10 566278 0.25 8.93-9-3 Deoxy/Methyl phosphonate ekk kke 10 566279 0.38 — 3-9-3Deoxy/Methyl phosphonate ekk kke 10 566280 0.27 47 3-9-3 Deoxy/Methylphosphonate ekk kke 10 566283 0.8 >100 3-9-3 Deoxy/Methyl phosphonateekk kke 10 573815 0.16 18.8 3-9-3 Deoxy/Phosphonoacetate ekk kke 10573816 0.55 18.1 3-9-3 Deoxy/Phosphonoacetate ekk kke 10 573817 0.1722.5 3-9-3 Deoxy/Phosphonoacetate ekk kke 10 573818 0.24 13.5 3-9-3Deoxy/Phosphonoacetate ekk kke 10 e = 2′-MOE, k = cEt

Example 83 Modified Oligonucleotides Comprising Methyl PhosphonateInternucleoside Linkages Targeting PTEN and SRB-1—In Vivo Study

Additional modified oligonucleotides were designed based on ISIS 482050and 449093 wherein the gap region contains tenβ-D-2′-deoxyribonucleosides. The modified oligonucleotides were designedby introducing two methyl phosphonate internucleoside linkages at the5′-end of the gap region with a 3/10/3 motif. The oligonucleotides wereevaluated for reduction in PTEN and SRB-1 mRNA expression levels invivo. The parent gapmers, ISIS 482050 and 449093 were included in thestudy for comparison.

The modified oligonucleotides and their motifs are described in Table127. Each internucleoside linkage is a phosphorothioate (P═S) except forthe internucleoside linkage having a subscript “x”. Each nucleosidefollowed by a subscript “x” indicates a methyl phosphonateinternucleoside linkage (—P(CH₃)(═O)—). Nucleosides followed by asubscript “d” is a β-D-2′-deoxyribonucleoside. Nucleosides followed by asubscript “k” indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt).“^(m)C” indicates a 5-methyl cytosine modified nucleoside.

Treatment

Six week old BALB/C mice (purchased from Charles River) were injectedsubcutaneously twice a week for three weeks at dosage 10 mg/kg or 20mg/kg with the modified oligonucleotides shown below or with salinecontrol. Each treatment group consisted of 3 animals. The mice weresacrificed 48 hrs following last administration, and organs and plasmawere harvested for further analysis.

mRNA Analysis

Liver tissues were homogenized and mRNA levels were quantitated usingreal-time PCR and normalized to RIBOGREEN as described herein. Theresults in Table 128 are listed as PTEN or SRB-1 mRNA expression foreach treatment group relative to saline-treated control (% UTC). Asillustrated, reduction in PTEN or SRB-1 mRNA expression levels wasachieved with the oligonucleotides comprising two methyl phosphonateinternucleoside linkages at the 5′-end of the gap region, ISIS 582073and 582074.

Plasma Chemistry Markers

Plasma chemistry markers such as liver transaminase levels, alanineaminotranferase (ALT) in serum were measured relative to saline injectedmice and the results are presented in Table 128. Treatment with theoligonucleotides resulted in reduction in ALT level compared totreatment with the parent gapmer, ISIS 482050 or 449093. The resultssuggest that introduction of methyl phosphonate internucleosidelinkage(s) can be useful for reduction of hepatoxicity profile ofotherwise unmodified parent gapmers.

Body and Organ Weights

Body weights, as well as liver, kidney and spleen weights were measuredat the end of the study. The results below are presented as the averagepercent of body and organ weights for each treatment group relative tosaline-treated control. As illustrated in Table 129, treatment with ISIS582073 resulted in a reduction in liver and spleen weights compared totreatment with the parent gapmer, ISIS 482050. The remainingoligonucleotide, ISIS 582074 did not cause any changes in body and organweights outside the expected range as compared to ISIS 449093.

TABLE 127Modified oligonucleotides comprising methyl phosphonate internu-cleoside linkages Wing SEQ ISIS Gap Chemistry ID NO Sequence (5′ to 3′)Motif Chemistry 5′ 3′ NO. 482050 A_(k)T_(k)^(m)C_(k)A_(d)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d)A_(d)G_(d)^(m)C_(k)T_(k)T_(k) 3-10-3 Full deoxy kkk kkk 85 582073 A_(k)T_(k)^(m)C_(k)A_(dx)T_(dx)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d)A_(d)G_(d)^(m)C_(k)T_(k)T_(k) 3-10-3 Deoxy/Methyl kkk kkk 85 phosphonate 449093T_(k)T_(k) ^(m)C_(k)A_(d)G_(d)T_(d) ^(m)C_(d)A_(d)T_(d)G_(d)A_(d)^(m)C_(d)T_(d)T_(k) ^(m)C_(k) ^(m)C_(k) 3-10-3 Full deoxy kkk kkk 86582074 T_(k)T_(k) ^(m)C_(k)A_(dx)G_(dx)T_(d)^(m)C_(d)A_(d)T_(d)G_(d)A_(d) ^(m)C_(d)T_(d)T_(k) ^(m)C_(k) ^(m)C_(k)3-10-3 Deoxy/Methyl kkk kkk 86 phosphonate k = cEt

TABLE 128 Effect of modified oligonucleotide treatment on targetreduction and liver function in BALB/C mice ISIS Dosage % ALT Gap WingChemistry SEQ NO. Target (mg/kg/wk) UTC (IU/L) Motif Chemistry 5′ 3′ IDNO. Saline — 0 100 30 — — — — — 482050 PTEN 10 50 228 3-10-3 Full deoxykkk kkk 85 482050 20 36.1 505 582073 10 72.2 47.7 Deoxy/Methyl kkk kkk85 582073 20 57.4 46 phosphonate 449093 SRB-1 10 48 543 3-10-3 Fulldeoxy kkk kkk 86 449093 20 18.5 1090 582074 10 51.3 58.3 Deoxy/Methylkkk kkk 86 582074 20 30.3 126.3 phosphonate k = cEt

TABLE 129 Effect of modified oligonucleotide treatment on body and organweights in BALB/C mice ISIS Dosage Body wt rel to Liver/Body Spleen/BodyKidney/Body SEQ NO. Target (mg/kg/wk) predose (%) Wt (%) Wt (%) Wt (%)ID NO. Saline — 0 108.4 100 100 100 482050 PTEN 10 107.4 154.9 141.8115.7 85 482050 20 111.3 176.7 142.3 112.5 582073 10 108.9 122.9 111.7100.0 85 582073 20 107.9 133.8 114.6 102.9 449093 SRB-1 10 101.3 105.9117.9 89.3 86 449093 20 95.3 118.6 129.6 93.0 582074 10 107.1 92.2 116.489.2 86 582074 20 103.8 95.5 128.8 91.9

Example 84 Modified Oligonucleotides Comprising Methyl PhosphonateInternucleoside Linkages Targeting Target-Y—In Vivo Study

Additional modified oligonucleotides were designed in the same manner asthe antisense oligonucleotides described in Example 24, wherein twomethyl phosphonate internucleoside linkages are introduced at the 5′-endof the gap region. The modified oligonucleotides were designed based onISIS 464917, 465178, 465984 and 466456 with a 3/10/3 motif. Theoligonucleotides were evaluated for reduction in Target-Y mRNAexpression levels in vivo. The parent gapmers, ISIS 464917, 465178,465984 and 466456 were included in the study for comparison.

The modified oligonucleotides and their motifs are presented in Table130. Each internucleoside linkage is a phosphorothioate (P═S) except forthe internucleoside linkage having a subscript “x”. Each nucleosidefollowed by a subscript “x” indicates a methyl phosphonateinternucleoside linkage (—P(CH₃)(═O)—). Each nucleoside followed by asubscript “d” is a β-D-2′-deoxyribonucleoside. Nucleosides followed by asubscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside.Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH₃ bicyclicnucleoside (e.g. cEt). “N” indicates modified or naturally occurringnucleobases (A, T, C, G, U, or 5-methyl C).

Treatment

Six week old BALB/C mice (purchased from Charles River) were injectedsubcutaneously twice a week for three weeks at dosage 10 mg/kg or 20mg/kg with the modified oligonucleotides shown below or with salinecontrol. Each treatment group consisted of 3 animals. The mice weresacrificed 48 hrs following last administration, and organs and plasmawere harvested for further analysis.

mRNA Analysis

Liver tissues were homogenized and mRNA levels were quantitated usingreal-time PCR and normalized to RIBOGREEN as described herein. Theresults below are listed as Target-Y mRNA expression for each treatmentgroup relative to saline-treated control (% UTC). As illustrated inTable 131, reduction in Target-Y mRNA expression levels was achievedwith the oligonucleotides comprising two methyl phosphonateinternucleoside linkages at the 5′-end of the gap region, ISIS 582071,582072, 582069 and 582070.

Plasma Chemistry Markers

Plasma chemistry markers such as liver transaminase levels, alanineaminotranferase (ALT) in serum were measured relative to saline treatedmice and the results are presented in Table 131. Treatment with theoligonucleotides resulted in reduction in ALT level compared totreatment with the parent gapmer, ISIS 464917, 465178, 465984 or 466456.The results suggest that introduction of methyl phosphonateinternucleoside linkage(s) can be useful for reduction of hepatoxicityprofile of otherwise unmodified parent gapmers.

Body and Organ Weights

Body weights, as well as liver, kidney and spleen weights were measuredat the end of the study. The results in Table 132 are presented as theaverage percent of body and organ weights for each treatment grouprelative to saline-treated control. As illustrated, treatment with ISIS582070 resulted in a reduction in liver and spleen weights compared totreatment with the parent gapmer, ISIS 466456. An increase in body andorgan weights was observed for ISIS 582071 as compared to ISIS 464917.The remaining oligonucleotides, ISIS 582072 and 582069 did not cause anychanges in body and organ weights outside the expected range as comparedto ISIS 465178 and 465984.

TABLE 130Modified oligonucleotides comprising methyl phosphonate internu-cleoside linkages Wing SEQ ISIS Gap Chemistry ID NO Sequence (5′ to 3′)Motif Chemistry 5′ 3′ NO. 464917N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k)N_(k)N_(k)3-10-3 Full deoxy kkk kkk 6 582071N_(k)N_(k)N_(k)N_(dx)N_(dx)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k)N_(k)N_(k)3-10-3 Deoxy/Methyl kkk kkk phosphonate 465178N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k)N_(k)N_(k)3-10-3 Full deoxy kkk kkk 6 582072N_(k)N_(k)N_(k)N_(dx)N_(dx)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k)N_(k)N_(k)3-10-3 Deoxy/Methyl kkk kkk phosphonate 465984N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(e)N_(e)N_(e)3-10-3 Full deoxy kkk eee 6 582069N_(k)N_(k)N_(k)N_(dx)N_(dx)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k)N_(k)N_(k)3-10-3 Deoxy/Methyl kkk kkk phosphonate 466456N_(k)N_(d)N_(k)N_(d)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(e)N_(e)5-9-2 or Full deoxy or kdkdk ee 6 3-11-2 deoxy/cEt or kdk 582070N_(k)N_(d)N_(k)N_(dx)N_(dx)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(e)N_(e)3-11-2 Deoxy/Methyl kdk ee phosphonate e = 2′-MOE, k = cEt, d =2′-deoxyribonucleoside

TABLE 131 Effect of modified oligonucleotide treatment on Target-Yreduction and liver function in BALB/C mice ISIS Dosage % ALT Gap WingChemistry NO. (mg/kg/wk) UTC (IU/L) Motif Chemistry 5′ 3′ Saline 0 10030 — — — — 464917 10 29 1244 3-10-3 Full deoxy kkk kkk 464917 20 30.12335 582071 20 10.2 274 3-10-3 Deoxy/Methyl kkk kkk phosphonate 46517810 4.9 1231 3-10-3 Full deoxy kkk kkk 465178 20 10.6 6731 582072 10 36.744.7 3-10-3 Deoxy/Methyl kkk kkk 582072 20 23.6 43.7 phosphonate 46598410 4.7 61 3-10-3 Full deoxy kkk eee 465984 20 0.9 57 582069 10 11.1 39.73-10-3 Deoxy/Methyl kkk kkk 582069 20 3.3 27.7 phosphonate 466456 10 9.5692 5-9-2 or Full deoxy or kdkdk ee 466456 20 10.5 2209 3-11-2 deoxy/cEtor kdk 582070 10 73.9 24 3-11-2 Deoxy/Methyl kdk ee 582070 20 51.3 36.7phosphonate e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

TABLE 132 Effect of modified oligonucleotide treatment on body and organweights in BALB/C mice Body Liver/ Spleen/ Kidney/ Dosage wt rel to BodyBody Body ISIS NO. (mg/kg/wk) predose (%) Wt (%) Wt (%) Wt (%) Saline 0108 100 100 100 464917 10 92.9 125 106.2 102.3 464917 20 71.1 110.9 67.2107.3 582071 20 104.6 135.2 142.8 89.8 465178 10 94.9 131.3 108.1 85.3465178 20 79.5 147.5 112 95.3 582072 10 109.2 117.3 111.7 104.8 58207220 107.1 130.1 107.2 99.8 465984 10 111.4 117.6 110.1 98.8 465984 20111.3 122.6 134.5 96.1 582069 10 107.8 106.2 97 100.6 582069 20 105.4115.8 106.2 100.4 466456 10 109.7 148.6 198.7 105.9 466456 20 101.2182.3 213.7 101.9 582070 10 111.2 100.3 116.7 100.8 582070 20 111.1108.9 115.6 95.7

Example 85 Short-Gap Chimeric Oligonucleotides Targeting Target-Y

A series of chimeric antisense oligonucleotides was designed based onISIS 464917 or 465178, wherein the central gap region contains ten2′-deoxyribonucleosides. These gapmers were designed by introducing2′-MOE modified nucleoside(s) at the wing(s) and/or shortening thecentral gap region to nine, eight, or seven 2′-deoxyribonucleosides.

The gapmers and their motifs are described in Table 133. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt).

TABLE 133 Short-gap antisense oligonucleotides targeting Target-Y SEQISIS ID NO Sequence (5′ to 3′) Motif NO. 464917N_(k)N_(k)N_(k)NNNNNNNNNNN_(k)N_(k)N_(k) 3-10-3 6 (kkk-d10-kkk) 465977N_(k)N_(k)N_(k)NNNNNNNNNNN_(e)N_(e)N_(e) 3-10-3 6 (kkk-d10-eee) 573331N_(e)N_(k)N_(k)NNNNNNNNNNN_(k)N_(k)N_(e) 3-10-3 6 (ekk-d10-kke) 573332N_(e)N_(e)N_(k)N_(k)NNNNNNNNNN_(k)N_(k)N_(e) 4-9-3 6 (eekk-d9-kke)573333 N_(e)N_(e)N_(e)N_(k)N_(k)NNNNNNNNN_(k)N_(k)N_(e) 5-8-3 6(eeekk-d8-kke) 573334N_(e)N_(e)N_(e)N_(e)N_(k)N_(k)NNNNNNNN_(k)N_(k)N_(e) 6-7-3 6(eeeekk-d7-kke) 573335 N_(e)N_(k)N_(k)NNNNNNNNNN_(k)N_(k)N_(e)N_(e)3-9-4 6 (ekk-d9-kkee) 573336N_(e)N_(k)N_(k)NNNNNNNNN_(k)N_(k)N_(e)N_(e)N_(e) 3-8-5 6 (ekk-d8-kkeee)573361 N_(e)N_(k)N_(k)NNNNNNNN_(k)N_(k)N_(e)N_(e)N_(e)N_(e) 3-7-6 6(ekk-d7-kkeeee) 573338 N_(e)N_(e)N_(k)N_(k)NNNNNNNNN_(k)N_(k)N_(e)N_(e)4-8-4 6 (eekk-d8-kkee) 573339N_(e)N_(e)N_(e)N_(k)N_(k)NNNNNNNN_(k)N_(k)N_(e)N_(e) 5-7-4 6(eeekk-d7-kkee) 573340N_(e)N_(e)N_(k)N_(k)NNNNNNNN_(k)N_(k)N_(e)N_(e)N_(e) 4-7-5 6(eekk-d7-kkeee) 573779 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e)3-8-5 6 (kkk-d8-keeee) 573780N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6 (kkk-d8-keeee)573806 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6(kkk-d8-keeee) 573782 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e)3-8-5 6 (kkk-d8-keeee) 573783N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6 (kkk-d8-keeee)573784 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6(kkk-d8-keeee) 573785 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e)3-8-5 6 (kkk-d8-keeee) 573786N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6 (kkk-d8-keeee)573787 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6(kkk-d8-keeee) 465178 N_(k)N_(k)N_(k)NNNNNNNNNNN_(k)N_(k)N_(k) 3-10-3 6(kkk-d10-kkk) 466140 N_(k)N_(k)N_(k)NNNNNNNNNNN_(e)N_(e)N_(e) 3-10-3 6(kkk-d10-eee) 573341 N_(e)N_(k)N_(k)NNNNNNNNNNN_(k)N_(k)N_(e) 3-10-3 6(ekk-d10-kke) 573342 N_(e)N_(e)N_(k)N_(k)NNNNNNNNNN_(k)N_(k)N_(e) 4-9-36 (eekk-d9-kke) 573343 N_(e)N_(e)N_(e)N_(k)N_(k)NNNNNNNNN_(k)N_(k)N_(e)5-8-3 6 (eeekk-d8-kke) 573344N_(e)N_(e)N_(e)N_(e)N_(k)N_(k)NNNNNNNN_(k)N_(k)N_(e) 6-7-3 6(eeeekk-d7-kke) 573345 N_(e)N_(k)N_(k)NNNNNNNNNN_(k)N_(k)N_(e)N_(e)3-9-4 6 (ekk-d9-kkee) 573346N_(e)N_(k)N_(k)NNNNNNNNN_(k)N_(k)N_(e)N_(e)N_(e) 3-8-5 6 (ekk-d8-kkeee)573347 N_(e)N_(k)N_(k)NNNNNNNN_(k)N_(k)N_(e)N_(e)N_(e)N_(e) 3-7-6 6(ekk-d7-kkeeee) 573348 N_(e)N_(e)N_(k)N_(k)NNNNNNNNN_(k)N_(k)N_(e)N_(e)4-8-4 6 (eekk-d8-kkee) 573349N_(e)N_(e)N_(e)N_(k)N_(k)NNNNNNNN_(k)N_(k)N_(e)N_(e) 5-7-4 6(eeekk-d7-kkee) 573350N_(e)N_(e)N_(k)N_(k)NNNNNNNN_(k)N_(k)N_(e)N_(e)N_(e) 4-7-5 6(eekk-d7-kkeee) 573788 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e)3-8-5 6 (kkk-d8-keeee) 573789N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6 (kkk-d8-keeee)573790 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6(kkk-d8-keeee) 573791 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e)3-8-5 6 (kkk-d8-keeee) 573792N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6 (kkk-d8-keeee)573793 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6(kkk-d8-keeee) 573794 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e)3-8-5 6 (kkk-d8-keeee) 573795N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6 (kkk-d8-keeee)573796 N_(k)N_(k)N_(k)NNNNNNNNN_(k)N_(e)N_(e)N_(e)N_(e) 3-8-5 6(kkk-d8-keeee) 141923C_(e)C_(e)T_(e)T_(e)C_(e)CCTGAAGGTTC_(e)C_(e)T_(e)C_(e)C_(e) 5-10-5 9(neg control) (e5-d10-e5) e = 2′-MOE (e.g. e5 = eeeee), k = cEt, d =2′-deoxyribonucleoside

Example 86 Short-Gap Chimeric Oligonucleotides Targeting Target-Y—InVitro Study

Several short-gap chimeric oligonucleotides from Table 133 were selectedand evaluated for their effects on Target-Y mRNA in vitro. The parentgapmer, ISIS 464917 and 465178 were included in the study forcomparison. ISIS 141923 was used as a negative control.

The newly designed gapmers were tested in vitro. Primary mousehepatocytes at a density of 35,000 cells per well were transfected usingelectroporation with 0.0625, 0.25, 1, 4 and 16 μM concentrations ofchimeric oligonucleotides. After a treatment period of approximately 24hours, RNA was isolated from the cells and Target-Y mRNA levels weremeasured by quantitative real-time PCR. Primer probe set RTSXXXX wasused to measure mRNA levels. Target-Y mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis presented in Table 134 and was calculated by plotting theconcentrations of oligonucleotides used versus the percent inhibition ofTarget-Y mRNA expression achieved at each concentration, and noting theconcentration of oligonucleotide at which 50% inhibition of Target-YmRNA expression was achieved compared to the control. As illustrated inTable 134 and 135, several short-gap oligonucleotides showed comparableinhibition of Target-Y mRNA levels as compared to the parent gapmers,ISIS 464917 or 465178.

TABLE 134 Comparison of inhibition of Target-Y mRNA levels of short-gapoligonucleotides with ISIS 464917 IC₅₀ SEQ ID ISIS NO Motif (μM) NO.464917 3-10-3 0.5 6 (kkk-d10-kkk) 573331 3-10-3 0.5 6 (ekk-d10-kke)573332 4-9-3 0.6 6 (eekk-d9-kke) 573333 5-8-3 0.5 6 (eeekk-d8-kke)573335 3-9-4 0.4 6 (ekk-d9-kkee) 573336 3-8-5 0.5 6 (ekk-d8-kkeee)573361 3-7-6 0.6 6 (ekk-d7-kkeeee) 573340 4-7-5 2.3 6 (eekk-d7-kkeee)141923 5-10-5 >16 9 (neg control) (e5-d10-e5) e = 2′-MOE (e.g. e5 =eeeee), k = cEt, d = 2′-deoxyribonucleoside

TABLE 135 Comparison of inhibition of Target-Y mRNA levels of short-gapoligonucleotides with ISIS 465178 IC₅₀ SEQ ISIS NO Motif (μM) ID NO.465178 3-10-3 0.2 6 (kkk-d10-kkk) 573341 3-10-3 0.2 6 (ekk-d10-kke)573342 4-9-3 0.4 6 (eekk-d9-kke) 573345 3-9-4 0.2 6 (ekk-d9-kkee) 5733463-8-5 0.4 6 573348 (ekk-d8-kkeee) 0.5 6 573350 4-8-4 0.9 6(eekk-d8-kkee) 573806 4-7-5 0.8 6 (eekk-d7-kkeee) 573783 3-8-5 1.0 6(kkk-d8-keeee) 573784 3-8-5 1.3 6 (kkk-d8-keeee) 573785 3-8-5 1.0 6(kkk-8-keeee) 573792 3-8-5 0.5 6 (kkk-8-keeee) 573794 3-8-5 0.4 6(kkk-d8-keeee) 573795 3-8-5 0.5 6 (kkk-d8-keeee) 573796 3-8-5 0.8 6(kkk-d8-keeee) 141923 5-10-5 >16 6 (neg control) (e5-d10-e5) e = 2′-MOE(e.g. e5 = eeeee), k = cEt, d = 2′-deoxyribonucleoside

Example 87 Short-Gap Chimeric Oligonucleotides Targeting Target-Y—InVivo Study

Several short-gap oligonucleotides described in Example 85 were selectedand evaluated for efficacy in vivo and for changes in the levels ofvarious plasma chemistry markers targeting Target-Y. The parent gapmer,ISIS 464917 was included in the study for comparison.

Treatment

Six week male BALB/C mice (purchased from Charles River) were injectedsubcutaneously with a single dose of antisense oligonucleotide at 10mg/kg or 20 mg/kg or with saline control. Each treatment group consistedof 4 animals. The mice were sacrificed 96 hrs following lastadministration, and organs and plasma were harvested for furtheranalysis.

mRNA Analysis

Liver tissues were homogenized and mRNA levels were quantitated usingreal-time PCR and normalized to Cyclophilin A as described herein. Theresults below are listed as Target-Y mRNA expression for each treatmentgroup relative to saline-injected control (% UTC). As illustrated inTable 136, Target-Y mRNA expression levels were reduced in adose-dependent manner with the newly designed oligonucleotides.

Plasma Chemistry Markers

Plasma chemistry markers such as liver transaminase levels, alanineaminotranferase (ALT) in serum were measured relative to saline treatedmice and the results are presented in Table 136. Treatment with thenewly designed oligonucleotides resulted in reduction in ALT levelscompared to treatment with the parent gapmer, ISIS 464917. The resultssuggest that shortening the central gap region and introducing 2′-MOEmodified nucleoside(s) at the wing(s) can be useful for the reduction ofhepatoxicity profile of ISIS 464917.

Body and Organ Weights

Body weights, as well as liver, kidney and spleen weights were alsomeasured at the end of the study. The results showed that treatment withthe newly designed oligonucleotides did not cause any changes in bodyand organ weights outside the expected range as compared to ISIS 464917(data not shown).

TABLE 136 Effect of short-gap antisense oligonucleotide treatment onTarget-Y reduction and liver function in BALB/C mice Dosage % ALT SEQISIS NO (mg/kg/wk) UTC (IU/L) Motif ID NO. Saline 0 99 23 — 464917 1011.5 1834 3-10-3 6 20 5.1 8670 (kkk-d10-kkk) 573333 10 32.8 79 5-8-3 620 21.2 370 (eeekk-d8-kke) 573334 10 79.5 26 6-7-3 6 20 69.4 29(eeeekk-d7-kke) 573336 10 23.2 179 3-8-5 6 20 12.0 322 (ekk-d8-kkeee)573339 10 47.9 35 5-7-4 6 20 32.8 199 (eeekk-d7-kkee) 573340 10 81.3 634-7-5 6 20 66.2 33 (eekk-d7-kkeee) 573361 10 33.6 150 3-7-6 6 20 19.2722 (ekk-d7-kkeeee) 573783 10 16.5 734 3-8-5 6 20 6.3 1774(kkk-d8-keeee) 573785 10 20.2 61 3-8-5 6 20 14.2 40 (kkk-d8-keeee)573806 10 19.3 346 3-8-5 6 20 15.4 1389 (kkk-d8-keeee) e = 2′-MOE, k =cEt, d = 2′-deoxyribonucleoside

Example 88 Short-Gap Chimeric Oligonucleotides Targeting PTEN

A series of chimeric antisense oligonucleotides was designed based onISIS 482050, wherein the central gap region contains ten2′-deoxyribonucleosides. These gapmers were designed by introducing2′-MOE modified nucleoside(s) at the wing(s) and/or shortening thecentral gap region to nine, or eight 2′-deoxyribonucleosides.

The gapmers and their motifs are described in Table 137. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine nucleobases throughout each gapmer are5-methyl cytosines. Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or“k” are sugar modified nucleosides. A subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k”indicates a 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt).

TABLE 137 Short-gap antisense oligonucleotides  targeting PTEN SEQ ISISID NO. Sequence (5′ to 3′) Motif NO. 482050A_(k)T_(k)C_(k)ATGGCTGCAGC_(k)T_(k)T_(k) 3-10-3 85 (kkk-d10-kkk) 508033A_(k)T_(k)C_(k)ATGGCTGCAGC_(e)T_(e)T_(e) 3-10-3 85 (kkk-d10-eee) 573351A_(e)T_(k)C_(k)ATGGCTGCAGC_(k)T_(k)T_(e) 3-10-3 85 (ekk-d10-kke) 573352A_(e)T_(e)C_(k)A_(k)TGGCTGCAGC_(k)T_(k)T_(e) 4-9-3 85 (eekk-d9-kke)573353 A_(e)T_(e)C_(e)A_(k)T_(k)GGCTGCAGC_(k)T_(k)T_(e) 5-8-3 85(eeekk-d8-kke) 573354A_(e)T_(e)C_(e)A_(e)T_(k)G_(k)GCTGCAGC_(k)T_(k)T_(e) 6-7-3 85(eeeekk-d7-kke) 573355 A_(e)T_(k)C_(k)ATGGCTGCAG_(k)C_(k)T_(e)T_(e)3-9-4 85 (ekk-d9-kkee) 573356A_(e)T_(k)C_(k)ATGGCTGCA_(k)G_(k)C_(e)T_(e)T_(e) 3-8-5 85 (ekk-d8-kkeee)573357 A_(k)T_(k)C_(k)ATGGCTGC_(k)A_(k)G_(e)C_(e)T_(e)T_(e) 3-7-6 85(ekk-d7-kkeeee) 573358 A_(e)T_(e)C_(k)A_(k)TGGCTGCAG_(k)C_(k)T_(e)T_(e)4-8-4 85 (eekk-d8-kkee) 573359A_(e)T_(e)C_(e)A_(k)T_(k)GGCTGCAG_(k)C_(k)T_(e)T_(e) 5-7-4 85(eeekk-d7-kkee) 573360A_(e)T_(e)C_(k)A_(k)TGGCTGCA_(k)G_(k)C_(e)T_(e)T_(e) 4-7-5 85(eekk-d7-kkeee) 573797 T_(k)G_(k)G_(k)CTGCAGCTT_(k)C_(e)C_(e)G_(e)A_(e)3-8-5 87 (kkk-d8-keeee) 573798A_(k)T_(k)G_(k)GCTGCAGCT_(k)T_(e)C_(e)C_(e)G_(e) 3-8-5 88 (kkk-d8-keeee)573799 C_(k)A_(k)T_(k)GGCTGCAGC_(k)T_(e)T_(e)C_(e)C_(e) 3-8-5 89(kkk-d8-keeee) 573800 T_(k)C_(k)A_(k)TGGCTGCAG_(k)C_(e)T_(e)T_(e)C_(e)3-8-5 90 (kkk-d8-keeee) 573801A_(k)T_(k)C_(k)ATGGCTGCA_(k)G_(e)C_(e)T_(e)T_(e) 3-8-5 85 (kkk-d8-keeee)573802 C_(k)A_(k)T_(k)CATGGCTGC_(k)A_(e)G_(e)C_(e)T_(e) 3-8-5 91(kkk-d8-keeee) 573803 C_(k)C_(k)A_(k)TCATGGCTG_(k)C_(e)A_(e)G_(e)C_(e)3-8-5 92 (kkk-d8-keeee) 573804T_(k)C_(k)C_(k)ATCATGGCT_(k)G_(e)C_(e)A_(e)G_(e) 3-8-5 93 (kkk-d8-keeee)573805 T_(k)T_(k)C_(k)CATCATGGC_(k)T_(e)G_(e)C_(e)A_(e) 3-8-5 94(kkk-d8-keeee) e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 89 Short-Gap Chimeric Oligonucleotides Targeting PTEN—In VitroStudy

Several short-gap chimeric oligonucleotides from Table 137 were selectedand evaluated for their effects on PTEN mRNA in vitro. The parentgapmer, ISIS 482050 were included in the study for comparison. ISIS141923 was used as a negative control.

The newly designed gapmers were tested in vitro. Primary mousehepatocytes at a density of 35,000 cells per well were transfected usingelectroporation with 0.0625, 0.25, 1, 4 and 16 μM concentrations ofchimeric oligonucleotides. After a treatment period of approximately 24hours, RNA was isolated from the cells and PTEN mRNA levels weremeasured by quantitative real-time PCR. Primer probe set RTS186 was usedto measure mRNA levels. PTEN mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotidewas calculated in the same manner as described previously and theresults are presented in Table 138. As illustrated, most short-gapoligonucleotides showed comparable inhibition of PTEN mRNA levels ascompared to ISIS 482050.

TABLE 138 Comparison of inhibition of PTEN mRNA levels of short-gapoligonucleotides with ISIS 482050 IC₅₀ SEQ ISIS NO Motif (μM) ID NO.482050 3-10-3 1.9 85 (kkk-d10-kkk) 573351 3-10-3 2.8 85 573353(ekk-d10-kke) 6.1 85 573355 3-9-4 2.6 85 (ekk-d9-kkee) 573798 3-8-5 1.688 (kkk-d8-keeee) 573799 3-8-5 1.9 89 (kkk-d8-keeee) 573803 3-8-5 1.4 92(kkk-d8-keeee) 141923 5-10-5 >16 9 (neg control) (e5-d10-e5) e = 2′-MOE(e.g. e5 = eeeee), k = cEt, d = 2′-deoxyribonucleoside

1.-272. (canceled)
 273. A oligomeric compound comprising a modifiedoligonucleotide consisting of 10 to 30 linked nucleosides, wherein themodified oligonucleotide has a modification motif comprising: a5′-region consisting of 2-8 linked 5′-region nucleosides, eachindependently selected from among a modified nucleoside and anunmodified deoxynucleoside, provided that at least one 5′-regionnucleoside is a modified nucleoside and wherein the 3′-most 5′-regionnucleoside is a modified nucleoside; a 3′-region consisting of 2-8linked 3′-region nucleosides, each independently selected from among amodified nucleoside and an unmodified deoxynucleoside, provided that atleast one 3′-region nucleoside is a modified nucleoside and wherein the5′-most 3′-region nucleoside is a modified nucleoside; and a centralregion between the 5′-region and the 3′-region consisting of 6-12 linkedcentral region nucleosides, each independently selected from among: amodified nucleoside and an unmodified deoxynucleoside, wherein the5′-most central region nucleoside is an unmodified deoxynucleoside andthe 3′-most central region nucleoside is an unmodified deoxynucleoside;wherein the modified oligonucleotide has a nucleobase sequencecomplementary to the nucleobase sequence of a target region of a targetnucleic acid.
 274. The oligomeric compound of claim 273, wherein the5′-region has a motif selected from among: AB, ABB, AAA, BBB, BBBAA,AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB,ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW,AAAAA, and BBBBAA; wherein the 3′-region has a motif selected fromamong: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB,BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA,AABBBB, BAAAA, and ABBBB; wherein the central region has a nucleosidemotif selected from among: DDDDDD, DDDDDDD, DDDDDDDD, DDDDDDDDD,DDDDDDDDDD, DDDDDDDDD, DXDDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD,DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXXDDDDDD, DDDDDDXXD, DDXXDDDDD,DDDXXDDDD, DDDDXXDDD, DDDDDXXDD, DXDDDDDXD, DXDDDDXDD, DXDDDXDDD,DXDDXDDDD, DXDXDDDDD, DDXDDDDXD, DDXDDDXDD, DDXDDXDDD, DDXDXDDDD,DDDXDDDXD, DDDXDDXDD, DDDXDXDDD, DDDDXDDXD, DDDDXDXDD, and DDDDDXDXD,DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD,DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD, DDXDXDDD,DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD,and DDDDDXXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD,DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD,and DDDDXXD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD,DDXXDD, DDXDXD, and DDDXXD; and wherein each A is a modified nucleosideof a first type, each B is a modified nucleoside of a second type, eachW is a modified nucleoside of a first type, a second type, or a thirdtype, each D is an unmodified deoxynucleoside, and each X is a modifiednucleoside or a modified nucleobase.
 275. The oligomeric compound ofclaim 274, wherein the 5′-region has a motif selected from among: AB,ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, ABBW, ABBWW, ABBB, ABBBB,ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB,BBBBB, AAABW, and BBBBAA; and wherein the 3′-region has a BBA motif.276. The oligomeric compound of claim 274, wherein each A nucleosidecomprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA,α-LNA, ENA and 2′-thio LNA.
 277. The oligomeric compound of claim 274,wherein each A nucleoside comprises a cEt.
 278. The oligomeric compoundof claim 276, wherein each B nucleoside comprises a 2′-substituted sugarmoiety comprising a 2′-substituent selected from among: a halogen, OCH₃,OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂, and OCH₂—N(H)—C(═NH)NH₂.
 279. Theoligomeric compound of claim 278, wherein each B nucleoside comprises a2′-substituted sugar moiety comprising a 2′-substituent selected fromamong: F, OCH₃, O(CH₂)₂—OCH₃.
 280. The oligomeric compound of claim 274,wherein one of A or B comprises a bicyclic sugar moiety, another of A orB comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidinenucleobase.
 281. The oligomeric compound of claim 274, wherein one of Aor B comprises a bicyclic sugar moiety, another of A or B comprises a2′-MOE sugar moiety, and W comprises FHNA.
 282. The oligomeric compoundof claim 274, wherein one of A or B comprises cEt, another of A or Bcomprises a 2′-modified sugar moiety, and W comprises a 2-thio-thymidinenucleobase.
 283. The oligomeric compound of claim 274, wherein one of Aor B comprises cEt, another of A or B comprises a 2′-modified sugarmoiety, and W comprises FHNA.
 284. The oligomeric compound of claim 274,wherein each A comprises MOE, each B comprises cEt, and each W isselected from among cEt or FHNA.
 285. The oligomeric compound of claim284, wherein each W comprises cEt.
 286. The oligomeric compound of claim284, wherein each W comprises 2-thio-thymidine.
 287. The oligomericcompound of claim 284, wherein each W comprises FHNA.
 288. Theoligomeric compound of claim 274 comprising at least one modifiedinternucleoside linkage.
 289. The oligomeric compound of claim 288,wherein each internucleoside linkage is a modified internucleosidelinkage.
 290. The oligomeric compound of claim 289 comprising at leastone phosphorothioate internucleoside linkage.
 291. The oligomericcompound of claim 288 comprising at least one methylphosphonateinternucleoside linkage.
 292. The oligomeric compound of claim 275,wherein each A nucleoside comprises a bicyclic sugar moiety selectedfrom among: cEt and LNA and each B nucleoside comprises a 2′-MOE.